All Hematology

GENERAL PRINCIPLES OF
ANAEMIA
Dr Kipkulei,
Moi University school of Medicine,
Dept. of Hematalogy&Blood Transfusion
TOPIC OUTLINE
• Definition of anaemia
• Aetiology of anaemia
• Physiological adaptations in anaemia
• Classification of anaemia
• Clinical features
• Laboratory evaluation of anaemia
• General principles of treatment
• Complications of anaemia
2
Definition
• Is a reduction of haemoglobin(Hb)
concentration below normal limits for a
given age and sex
3
Aetiology of anemia
1) Excessive blood loss

 Acute blood loss
 Chronic blood loss
2) Excessive destruction of red blood cells
(hemolysis)
 Hereditary- membrane defects, hemoglibinopathies,
enzymopathies
 Acquired- immunohemolytic, nonimmunohemolytic
4
3) Diminished production of red blood cells from
bone marrow
• Iron deficiency anemia
• Megaloblastic anemias
– Vitamin B12 deficiency; Pernicious anemia
– Folate deficiency
• Anemia of chronic disease
• Aplastic anemia
• Infiltration of bone marrow by malignant cells
• Pure red cell aplasia
5
Physiological adaptations in anemia
• Generally anaemia leads to tissue hypoxia.
• Tissue hypoxia initiates physiologic compensatory
mechanisms, which include:
1.Increase in conc. of 2,3-diphosphoglycerate leading
to increase in release of O2 from red cells to the
tissues (shifting of the 02-dissociation curve to
the right)
2.Increase in cardiac output
3.Reduction in peripheral vascular resistance-( occurs
when the anaemia is fairly severe-Hb is <7gm/dl)
6
Physiologic adaptations….
4.Increase in rate and depth of breathing
5.When anaemia becomes more severe, there is
redistribution of blood flow from ‘non vital’
organs, e.g. kidney&skin to vital organs e.g. brain
and heart
6.Decreased renal blood flow in turn causes an
activation of renin-angiotensin system, leading to
salt and water retention(leading to increased
plasma voume)
7.Increase in the production of erythropoietin
7
Classification of anemia
• Pathological classification is based on:
• Aetiology e.g. iron deficiency anemia, hemolytic
anemia , aplastic anemia etc
• Morphology(RBC size and colour)
• Clinical classification is based on:
• Rapidity of onset(acute or chronic) and
• Severity(level of Hb and clinical features)
8
Classification….
Classification based on morphology: RBC
size(MCV) and colour
i. Normocytic normochromic (MCV 76-96fl and
normal colour): BM failure, Aplastic
anaemia, Myelofibrosis,
Leukemia/metastasis, renal failure, anaemia
of chronic disease, anaemia of acute blood
loss, hemolytic anaemias
9
Classification….
ii. Microcytic normochromic (MCV <76fl and
normal color): Anemia of chronic disease,
sideroblastic anaemia, lead poisoning etc
iii. Macrocytic normochromic (MCV >96fl and
normal color): Megalolastic anaemia(vit B12
and folate deficiency), alcoholic liver disease
etc
10
Classification….
iv. Microcytic hypochromic (MCV <76 and decrease in
RBC color): Iron Deficiency Anemia, thalassemia,
anaemia of chronic disease(rare cases)
NB: Reticulocyte count is also used to classify
normocytic anaemias:
a) Those associate low retic count: BM failure,
aplastic anaemia, myefibrosis, leukemia/mets
b) Normocytic anaemias associated with high retic
count: hemolytic anaemias, acute blood loss
11
Classification…
Clinical classification based on the severity (level of Hb) -WHO
Severity classification
Haemoglobin (Hb)
Grade 1 (Mild) 10-11.9 gm/dl
Grade 2 (Moderate) 7-9.9 gm/dl
Grade 3 (severe) < 7 gm/dl
12
Clinical features- signs&symptoms
• The s&s can be related to the anaemia itself or the
underlying cause
• The s&s depends on:
– Severity of anaemia
– Rapidity of onset
– Demands of physical activity
– Presence of intercurrent illness
– Age of patient- less tolerated in elderly
13
Clinical features….
Symptoms:
• General symptoms include weakness/fatigue,
general malaise, lethargy, dizziness, headache,
shortness of breath, dyspnoea on exertion,
palpitations, syncope and symptoms of heart failure
• Specific symptoms related to the underlying cause
e.g. pica and restless legs syndrome in IDA etc
14
Clinical features….
Signs:
• General signs include pallor of skin, mucasal linings
and nail bed; tachycardia; flow(haemic) murmurs;
orthostatic hypotension,cardiomegally and signs of
heart failure.
• Specific signs of underlying cause e.g. koilonychia(in
IDA), jaundice(in hemolytic anaemia), bone
deformities(in thalassaemia), leg ulcers(in SCA)
15
Laboratory evaluation
i) Complete blood count
 Total red blood cell count
 Hb level
 Hematocrit/PCV
 MCV, RDW, MCH, MCHC
 Total WBC and differentials
 Platelet count
16
Laboratory evaluation(cont.)
ii) Peripheral blood film- morphology of RBCs
 Size- normocytic, macrocytic, microcytic
 Shape- e,g spherocytes, sickle cell, etc
 Colour- hypochromasia(e.g. in IDA),
hyperchromasia(e.g. in megaloblastic
anaemia, spherocytosis), polychromasia(in
hemolytic anaemias)
 Inclusions e.g. malaria parasites, Heinz
bodies etc
17
iii) Reticulocyte count
 Normal 2%
 Count <1%- indicate inadequate BM
production
 Count >4%- indicate hemolysis or acute
blood loss
vi. Bone marrow examination-biopsy or
aspiration
18
iv) Tests to rule out or support suspected
underlying cause e.g. ferritin, serum iron
and trasnferrin(IDA), RBC folate and vitamin
B12 level(megaloblastic anaemia), Sickling
test and Hb electrophoresis(SCA), renal
function tests(chronic renal failure)
19
Complications of anemia
1) Chronic anaemia may lead to impaired neurogical
development, leading to behavioral disturbances in
infants and reduced academic performance in
children
2) Worsening of preexisting clinical
conditions/intercurrent illnesses e.g. cardiovascular
disease
3) Heart failure
4) Death
20
Principles of treatment
Supportive
• blood transfusion- packed red cells
• Management of complications e.g. oxygen
therapy, antifailure drugs etc
Definitive
• Treatment of underlying cause of anemia e.g.
– Iron supplementation in iron deficiency anemia
– Vitamin B12 and folic acid in megaloblastic
anemia
21
IRON DEFICIENCY ANAEMIA
Dr. Kipkulei C.J

Department of haematology and
Blood Transfusion
22
TOPIC OUTLINE
• Overview of iron metabolism
• Definition
• Aetiology
• Pathogenesis
• Classfication
• Clinical features- symptoms and signs
• Laboratory features/diagnosis
• Treatment
• Other causes of microcytic anaemias
23
IRON DEFICIENCY ANEMIA-
Iron metabolism
• Iron is required for the synthesis of

haemoglobin, which is the main protein of
the red blood cell
• Its absorption is in duodenum
• Transferrin transports iron to the cells
• Ferrritin and Hemosiderin store iron
• 10% of daily iron is absorbed
24
Iron metabolism….
• Most body iron is present in hemoglobin in
circulating red cells
• The macrophages of the reticuloendotelial
system store iron released from
hemoglobin as ferritin and hemosiderin
• Small loss of iron each day in urine,
faeces, skin and nails and in menstruating
females as blood (1-2 mg daily)
25
Iron metabolism
• Iron concentration (Fe)-N: 60-170 mg/dl
• Total Iron Binding Capacity-N: 50-
450mg/dl
• Transferrin saturation-N: 20-50
• Ferritin concentration-N: 50-300 g/l
26
IRON DEFICIENCY ANAEMIA-
Definition
It is anaemia resulting from deficiency of iron
and characterized by microcytic and
hypochromic red blood cells
27
Aetiology
1.Chronic blood loss
• Menorrhagia
• Peptic ulcer
• Stomach cancer
• Ulcerative colitis
• Colorectal cancer
• Haemorhoids
• Hookworm infestation
2.Dietary insufficiency
3. Increased iron requirement- children, pregnancy, lactation
4. Malabsorption- gluten-induced enteropathy, gastrectomy,
atrophic gastritis, etc
28
Pathogenesis(stages)
• Prelatent
– reduction in iron stores without reduced serum iron levels
• Hb (N), MCV (N), iron absorption (), transferin saturation
(N), serum ferritin (), marrow iron ()
• Latent
– iron stores are exhausted, but the blood hemoglobin level
remains normal
• Hb (N), MCV (N), TIBC (), serum ferritin (), transferrin
saturation (), marrow iron (absent)
• Iron deficiency anemia
– blood hemoglobin concentration falls below the lower limit of
normal
• Hb (), MCV (), TIBC (), serum ferritin (), transferrin
saturation (), marrow iron (absent)
29
General symptoms/signs
• Fatigability
• Dizziness
• Headache
• Irritability
• Palpitations
• Pallor
• Signs and symptoms of Congestive HF
30
Characteristic symptoms/signs
• Glossitis, stomatitis
• Dysphagia(Plummer-Vincent syndrome)
• Atrophic gastritis
• Dry pale skin
• Koilonychia
• Blue sclerae
• Hair loss
• Pica
• Splenomegaly(in about 10%)
• Slight thrombocytosis
31
Koilonychia
32
Diagnosis
History and Physical exam-Symptoms and
sings to determine etiology
Laboratory investigations/findings:
 Complete Blood Count
• MCV ↓
• MCH ↓
• MCHC normal or ↓
• ↑RDW
• White blood cell count- normal
• Platelets may be↑
33
ANGULAR CHEILITIS AND SMOOTH TONGUE IN IRON
DEFICIENCY
34
Diagnosis
Laboratory investigations/findings(cont.)
Peripheral blood film
• Microcytosis
• Hypochromia
• Anisocytosis; red cells of unequal sizes
• Poikilocytosis( different shapes)- target
cells, elliptocytes
35
36
37
Diagnosis
Labortory investigations/findings(cont.)
Blood iron studies
• Iron levels ↓
• Totoal iron binding capacity(TIBC) ↑
• Transferrin saturation ↓
• Ferritin ↓
38
Diagnosis
Laboratory investigations/findings(cont,)
Bone marrow findings
• High cellularity
• Mild to moderate erythroid
hyperplasia(25-35%, N16-18%)
• Polychromatic and pyknotic cytoplasm
of erythroblasts
• Absence of stainable iron
39
Diagnosis
Laboratory investigations/findings(cont.)
Investigations to find out suspected
etiology e.g
• Colonoscopy
• Stool for ova and cysts of parasites
• Stool for occult blood
• OGD
• Gynecologic examinations
40
Treatment
1.Definitive- iron supplementation
• Oral iron
• Parenteral iron
2. Treatment of underlying cause
41
OTHER CAUSES OF MICROCYTIC
ANAEMIA
1. Chronic disease
2. Sideroblastic anaemia
3. Thalassemia
4. Lead poisoning
42
SIDEROBLASTIC ANEMIA
• Characterized by
– Increase in total body iron
– Presence of ringed sideroblasts in bone
marrow
– Hypochromic anemia
• Classification
– Hereditary form
– Acquired form(more common)
– Idiopathic- refractory anemia with ringed
sideroblasts- acquired stem disorder
– Secondary- lead poisoning, alcoholism,
malignancy 43
SIDEROBLASTIC ANEMIA-
Pathophysiology
• Disturbances of enzymes regulating
heme synthesis
• Ringed sideroblasts form when
nonferritin iron accumulated in the
mitochondria that circle the normoblast
nucleus
44
Laboratory findings
Peripheral Blood
• Moderate to severe anemia – dimorphic
(mild macrocytosis prevalent in RARS and
alcoholism)
• Target cells
• Pappenheimer bodies
• Basophilic stippling – coarse basophilic
stippling characteristic in lead poisoning
• ↓Fe, N to ↑TIBC, ↓% saturation, ↓ ferritin
45
Laboratory findings
Bone marrow
• Erythroid hyperplasia - megaloblastosis
• Ringed sideroblasts in 50% of
normoblasts
46
Therapy
• Pyroxidine – hereditary form (50%
response)
• Folic acid if megaloblastoid features
• Phlebotomy/chelation therapy to remove
excess iron
47
ANEMIA OF CHRONIC DISEASE
 Anemia that occurs in patients with:

– Chronic infections
– Chronic inflammatory disorders
– Neoplastic disorders
 Characterized by low serum iron, normal
iron stores
48
ANEMIA OF CHRONIC DISEASE-
Pathophysiology
 Impaired marrow response to anemia –

cytokines produced as result of immune
response inhibit erythropoietin production
 Block in iron release from macrophage
 Shortened erythrocyte survival
49
ANEMIA OF CHRONIC DISEASE-
Laboratory findings
 Mild anemia
 Normocytic, normochromic (some are
normocytic, hypochromic)
 ↓serum Fe
 Normal to ↑ TIBC
 Normal to ↑ transferrin saturation
 Normal or ↓ ferritin
50
MEGALOBLASTIC ANAEMIA
Dr. Kipkulei J.C

Department of Haematology and
Blood Transfusion
TOPIC OUTLINE
• General features
• Aetiology
• Laboratory features
• Treatment
• Pernicious anaemia
• Causes of macrocytic non-megaloblastic
anaemias
MEGALOBLASTIC ANEMIA-
General features
• Macrocytic type of anemia resulting from
impaired DNA synthesis due to mainly
deficiency of folate and or Vitamin
B12(Cobalamin)
• Slowly developing anemia, usually well
compensated
• Response to therapy rapidly and dramatic
• Treatment essential to avoid other
complications
Aetiology
• Major causes of Vitamin B12(Cobalamin)
deficiency
• Inadequate dietary intake- esp. vegetarians
• Malabsorption- autoimmune/ gastric atrophy
(Pernicious anemia), total gastrectomy,
Crohns’s disease, ileal resection, Tropical
sprue
• Competing organisms-Bacteria overgrowth in
blind loop syndrome and fish tapeworm
(Diphyllobothrium latum)
Aetiology
• Major causes of folate deficiency
• Poor dietary intake- e.g. alcoholism,
poverty, elderly, infants
• Malapsorption- tropical sprue, coeliac
disease, other small intestine disorders
• Increased requirements- e.g.pregnancy,
Severe hemolytic anemia
• Antifolate drugs- e.g. anticonvulsant
drugs(barbiturates, phenytoin, prmidone),
nitrofurantoin, tetracycline etc
Clinical features
Vitamin B12 deficiency

Peripheral manifestations
• General signs and symptoms of anemia
• Glossitis/stomatitis
Clinical features
Vitamin B12 deficiency(cont.)
Central manifestations
• Brain: Dementia, Psychological disturbances
• Spinal cord: Demyelinating disease,Loss of
posterior & lateral columns-"Combined
degneration of the cord"
• Neurologic disease stabilized with treatment,
but usually not reversed
• Treatment with folate DOES NOT
REVERSE the neurologic disease
Clinical features
Folate deficiency
• Peripheral manifestation- same as in
Vitamin B12 deficiency
• Central manifestations- NONE
Laboratory features
• Macrocytic normochromic anemia with trademark
oval macrocyte cell(MCV > 100 fl)
• Hypersegmented neutrophils - 98%
• Pancytopenia, esp if anemia severe
• Reticulocytopenia
• LDH elevated (90%)
• Serum Fe normal or elevated
• Serum B12 or folate low
• Bone Marrow shows intense erythroid hyperplasia
with abnormal morphology. Macroovalocytes and
occasional megaloblasts can be seen
PERNICIOUS ANEMIA
• Autoimmune destruction of parietal cells
• Antibodies against parietal cells, intrinsic factor
• Atrophic glossitis
• Chronic atrophic gastritis
• Achlorhydria is universal
• Increased incidence of gastric cancer
• Often associated with other immune diseases
(eg Hashimoto's thyroiditis)
PERNICIOUS ANEMIA
• Neurologic manifistations” (paresthesias,
numbness, weakness, memory loss,
personality changes, ataxia, loss of
vibration and position sense, psychosis
“megaloblastic madness’’)
• Deficient serum vitamin B 12 levels
• Schilling test
• Treatment: IM Vit B12 indefinitely
CAUSES OF MACROCYTIC
NORMOBLASTIC ANAEMIAS
• Chronic liver disease
• Alcoholism
• Hypothyroidism
• Myelodysplastic syndromes
• Pellagra
• Myxoedema and hypothyroidism
• Multiple myeloma, lymphoma, carcinomatosis
• Some drugs e.g. azathioprine
HAEMOLYTIC ANAEMIAS
Dr. Kipkulei C.J

Department of Haematology and
Blood Transfusion
TOPIC OUTLINE
• Definition
• Aetiologic classification
• Mechanism of haemolysis
• Laboratory features
• Treatment
• Sickle cell anaemia
• Thallasemias
HEMOLYTIC ANEMIAS
• Hemolytic anemia refers to↓RBCs due to
hemolysis(↓RBC life span)
RBC Components that Determine

Lifespan
• Membrane cytoskeleton
• ATP-generating pathway
• Hemoglobin
HEMOLYTIC ANEMIA-
Classification/Causes
• Causes of hemolytic anemis can be either
genetic/intracorpuscular or acquired/extracorpuscular
Genetic causes
• Genetic conditions of RBC membrane
– Hereditary spherocytosis
– Hereditary elliptocytosis
• Genetic conditions of RBC metabolism (enzyme defects)
– Glucose-6-phosphate dehydrogenase deficiency (G6PD
or favism)
– Pyruvate kinase deficiency
• Genetic conditions of hemoglobin
– Sickle cell anemia
– Thalassaemia
HEMOLYTIC ANEMIA-
CLASSIFICATION/CAUSES
Acquired
1.Immune mediated hemolytic anemia-direct Coombs test
is positive
Autoimmune hemolytic anemia
• Warm antibody (IgG) autoimmune hemolytic anemia
– Idiopathic
– Systemic lupus erythematosus (SLE)
– Evans' syndrome (antiplatelet antibodies and hemolytic antibodies)
• Cold antibody (IgM) autoimmune hemolytic anemia
– Idiopathic cold hemagglutinin syndrome
– Infectious mononucleosis and mycoplasma ( atypical) pneumonia
– Paroxysmal cold hemoglobinuria (rare)
HEMOLYTIC ANEMIA-
Acquired causes(cont.)
Immune mediated hemolytic anemia-direct Coombs
test is positive
Alloimmune hemolytic anemia
• Hemolytic disease of the newborn (HDN)
– Rh disease (Rh D)
– ABO hemolytic disease of the newborn
– Anti-Kell hemolytic disease of the newborn
– Rhesus c hemolytic disease of the newborn
– Other blood group incompatibility (RhC, Rhe, RhE, Kidd
antigen system, Duffy antigen, MN, P and others)
• Alloimmune hemolytic blood transfusion reactions (ie from a
non-compatible blood type)
• Drug induced immune mediated hemolytic anemia
– Penicillin (high dose)
– Methyldopa
HEMOLYTIC ANEMIA-
Acquired causes(cont.)
Non-immune mediated haemolytic anaemia-direct Coombs test
is negative
• Drugs (i.e., some drugs and other ingested substances lead to
hemolysis by direct action on RBCs)
• Toxins (e.g., snake venom)
• Trauma- Mechanical (heart valves, extensive vascular surgery,
microvascular disease)
• Microangiopathic hemolytic anemia (a specific subtype with
causes such as TTP( Thrombotic thrombocytopenic purpura), HUS(
Hemolytic uremic syndrome), DIC and HELLP( Hemolysis with
elevated liver function tests and low platelets) syndrome)
• Infections- e.g. Malaria, Babesiosis, septicaemia
• Membrane disorders e.g.Paroxysmal nocturnal
hemoglobinuria(PNH,rare acquired clonal disorder of red blood cell
surface proteins), liver disease
• Hypersplenism
HEMOLYTIC ANEMIA
Mechanisms of hemolysis
• Intravascular
• extravascular
HEMOLYTIC ANEMIA-
Mechanism of hemolysis
Intravascular hemolysis(IH)
• Rbc destruction occur in intravascular space
• Conditions associated with IH include:
– Acute hemolytic transfusion reaction
– Severe and extensive burns
– PNH
– Severe micorangiopathic hemolysis
– Physical trauma
– Sepsis
HEMOLYTIC ANEMIA-
Mechanism of hemolysis….
Extravascular hemolysis(EH)
• RBC destruction occur in the
reticuloendothelial system
• Conditions associated with EH
– Autoimmune hemolysis
– Delayed hemolytic transfusion reactions
– Hereditary spherocytosis
– Hypersplenism
– Hemolysis with liver disease
red cells destruction occurs in reticuloendothelial
system
- clinical states associated with extravascular hemolysis :
HEMOLYTIC ANEMIA-
Clinical features
• Symptoms and signs of anemia
• Jaundice
• Splenomegaly / hepatosplenomegaly
• Occassionally fever and tachycardia
HEMOLYTIC ANEMIA-
Laboratory investigations/findings
• Full blood count
– ↓RBC count
– ↑Reticulocyte count
• Peripheral blood smear/film
– Schistocytes
– Spherocytes
– Elevated number of reticulocytes
– Erythroblasts can be seen
– Heinz bodies
• Bone marrow
– erytrhroid hyperplasia (megaloblasts)
• Other blood tests/findings
– ↑unconjugated bilirubin in blood
– ↑LDH
HEMOLYTIC ANEMIA-
• Other blood tests/findings(cont.)
– ↓Haptoglobin and hemopexin levels
– ↑Iron levels
– Direct Coomb’s test positive in cases of
immune mediated hemolysis
– ↑Excretion of urobilinogen in the urine and
stercobilinogen in the stool
NB: Free hemoglobin(hemoglobinemia),

methemalbuminemia,hemoglobinuria&
hemosiderinuria indicates chronic
intravascular hemolysis.
HEMOLYTIC ANEMIA-
Treatment
• Compensated hemolysis – observation (clinical
evaluation) and folic acid at an oral dose
1mg/day
• Decompensated hemolysis
– Symptomatic treatment -blood transfusion: if
there is marked anemia
– Definitive therapy depends on the cause:
• In immune-related hemolytic anemia:
steroid therapy , immunosupressive agents,
immunoglobulins
• splenectomy can be helpful where
extravascular heamolysis is predominant
• Folic acid
SICKLE CELL ANEMIA-
Definition
• Chronic hemolytic anemia characterized
by sickle-shaped red cells(RBCs) caused
by homozygous inheritance of Hemoglobin
S(HbS)
SICKLE CELL ANEMIA-
Pathogenesis
- In HbS, valine is substituted for glutamic acid in
the sixth amino acid of the ß chain.
- Deoxy-HbS is much less soluble than deoxy HbA;
it forms a gelatinous network of fibrous polymers that
cause RBCs to sickle at sites of low pO2.
- Sickle RBCs are too fragile to withstand the
mechanical trauma of circulation→ Hemolysis
- Distorted, inflexible RBCs adhering to vascular
endothelium→Occlusion in micovascular circulation
SICKLE CELL ANEMIA-
Epidemiology
• Incidence of homozygous disease(SCA)
is lower than heterozygous disease(sickle
cell trait)
• In Kenya more prevalent in Nyanza,
Western and Coastal regions
SICKLE CELL ANEMIA-
Clinicsl features
1.Clinical complications due to severe hemolytic anaemia
- slowed growth and development in children
- bilirubins stones
- congestive heart failure from chronic anemias and
cardiac overload compensation
2.Consequences of vaso-occlusion of the microcirculations
(tissue ischemia and infarction)
- infarction of spleen(leads to autosplenectomy), brain,
marrow, kidney, lung, aseptic necrosis, central nervous
system and ophtalmic vascular lesions
3. Crises: painful vaso-occlusive crisis, visceral
sequestration crisis, aplastic crisis and hemolytic crisis
Factors which predispose to crises: dehydration, hypoxia,
acidosis, sepsis, hypothermia
SICKLE CELL ANEMIA-
• Full blood count
– Normocytic or slightly macrocytic anemia
– Leucocytosis(chronic neutrophilia)
– Mild thrombocytosis
– Reticulocytosis
• Peripheral blood film
– Sickle shaped cells
– Polychromasia
– Howell-Jolly bodies
• Other blood tests/findings
– Hb-electrophoresis
– Sickling test
SICKLE CELL ANEMIA-
Treatment
• Supportive measures
– Penicillin prophylaxis and pnuemococcal vaccination against
infections
– Proper hydration
– Analgesics and antipyretics for pain and fever
– Blood transfusions for very severe anemia
– Prevention hypoxemia and cold exposure
• New approaches to therapy;
– Activation of HbF synthesis: hydroxyurea, 5-azacytidine,
decytabine
– Antisickling agents acting on hemoglobin or membrane
(preclinical testing, clinical trials)
– Bone marrow transplantation
– Gene therapy
OTHER CAUSES OF NORMOCYTIC
ANAEMIAS
• Aplastic anaemia
• Acute haemorrhage
• Renal failure
• Myelophthisis- marrrow replacement by
fibrosis, tumor, or granulomatus disorders
THALASSAEMIAS
• Genetic disorders in which there is diminished
production of normal globin chains- most
often the α or β chains
Pathogenesis
• Are inherited as autosomal codominant; each
disorder may occur in a homozygous or
heterozygous form
Pathophysiology
• Due to diminished synthesis of one globin chain
there is relative excess of other globin chains
• Deficiency of globin chains leads to reduced
amount of hemoglobin and hence the red cell are
microcytic and hypochromic
• Excess globin chains may precipitate in the red
cell causing hemolysis, hence hemolytic anemia
Classification
• Alpha thalasaemia- absent or reduced synthesis
of α chains leading to reduced production of HbF,
HbA, HbA2 and excess β,γ,δ chains
– α Thalassaemia trait- two gene deletions(- -/αα OR
-α/-α)
– HbH disease- three gene deletions(- -/- α):
– α Thalassaemia major/Barts Hydrops syndrome- four
gene deletions( - -/- -): Lethal in utero
• Beta thalassaemia- absent or reduced
synthesis of β chains; reduced production of
HbA
– Thalassamia minor- βo/β or β+/β
– Thalassaemia major- βo/βo or β+/β+
βo Means absent gene

β+ Means defective gene
Geographical distribution
• Found most frequently in the
Mediterranean, Africa, Western and
Southeast Asia, India and Burma
• Distribution parallels that of Plasmodium
falciparum and the thalassaemia trait is
believed to offer resistance to falciparum
malaria
Beta thalassaemia major
• Severe microcytic hypochromic anemia

• Hepatosplenomegaly
• Prolonged marrow hyperplasia causes thickening
of the calvarium, giving mongoloid facies and a
‘hair on end’ appearance of the skull X-ray
• Reticulocytosis, nucleated red cells and target
cells on PBF
• Many patients die in infancy or childhood, while
those surviving longer develop severe
hemosiderosis due chronic blood transfusion
Beta thalassaemia major…
Laboratoy diagnosis
1. CBC- microcytic hypochromic anemia
2. PBF- raised reticulocytes, nucleated red cells
and target cells, poikilocytosis
3. Confirmatory test is measurement of the α/β
globin chain synthesis
4. Prenatal diagnosis is by globin chain analysis
on fetal blood
Beta thalassaemia major…
Treatment
Supportive
• Folate
• Chronic blood transfusion with chelation
therapy with desferrioxamine
• Splenectomy
Definitive
• Bone marrow transplantation
Benign/Reactive White blood
Cell Disorders
Dr.Lotodo T.L.C
Introduction
• Acquired disorders primarily involving white
cells may be either reactive or neoplastic.
• Neoplastic disorders result from the clonal
proliferation of a haemopoietic stem cell,
either myeloid or lymphoid, that has
undergone mutation.
Contents.
– Leucocytosis and leucopenia
– Neutrophilia and neutropenia
– Lymphocytosis and lymphopenia
– Monocytosis
– Eosinophilia
– Basophilia
– Infective Mononucleosis
LEUCOCYTES BENIGN DISORDERS
• Quantitative
– Change in number
– Terminology
– Cytosis / philia
» Increase in number
– Cytopenia
» Decrease in number
• Qualitative
– Morphologic changes
– Functional changes
Quantitative changes
Relative vs Absolute values

– Total white blood cell count
– Differential count
– Absolute count
• Differential gives the relative percentage of
each WBC
• Absolute value gives the actual number of each
WBC/mm3 of blood
Quantitative changes (LEUCOCYTOSIS)
• Leucocytes
– Phagocytes
• Granulocytes
– Neutrophils
– Eosinophils
– Basophils
• Mononuclear phagocytic cells
– Monocytes
– Macrophage and dendretic cells
– Lymphocytes
• B-cells
• T-cells
Quantitative changes (LEUCOCYTOSIS)
• Definition-Leukocytosis is an increased
number of white blood cells above the
normal range for age and sex, in the blood.
For adults this is > 11.0 x109/L leukocytes.
– Normal reference range (adult 21 years)
» 4.5 -- 11.0 x 109/L
Leucocytosis
• A leukocytosis is
frequently accompanied
by cytologic
abnormalities, such as
toxic granulation or
Dohle bodies.
Leukocytosis-Causes
• Infection
• Drugs- corticosteroids, antibiotics or anti-
seizure drugs
• Severe physical or emotional stress
• Chronic bone marrow diseases such as a
myeloproliferative disorder
• Acute or chronic leukemia
• Tissue damage, such as from burns
Creutzfeldt-Jakob Disease
Quantitative changes (LEUCOPENIA)
• Definition
TWBC lower than the reference range for the
age is defined as leucopenia
– Leucopenia may affect one or more lineages and it
is possible to be severely neutropenic or
lymphopenic without a reduction in total white
cell count.
Quantitative changes (NEUTROPHILIA)
• Definition
– Increase in the number of neutrophils and / or its precursors
for age
– In adults normal count is 2.0-7.5 x109/L(40-75%)
– Patients present with fever
– There is an increase in band form, metamyelocytes and
myelocytes (left shift)
– The film also shows toxic granulations,vacoulations and dohle
bodies(basophilic inclusions of denatured RNA in cytoplasm)
– NAP(neutrophil alkaline phosphatase)score is elevated-
intensity of staining of 100 neutrophils score as 0,1,2,3,4
Quantitative changes (NEUTROPHILIA) contd.
• Causes of Neutrophilia
– Infection
• Bacterial
– Inflammatory conditions
• Autoimmune disorders
• Gout
– Neoplasia
– Metabolic conditions
• Uraemia
• Acidosis
• Haemorhage
– Corticosteroids
– Marrow infiltration/fibrosis
– Myeloproliferative disorders
Quantitative changes (NEUTROPHILIA) contd.
• Chronic neutrophilia
• Long term corticosteroid
therapy
• Chronic inflammatory
reactions
• Infections or chronic
blood loss
Leukemoid reactions
• Applied to chronic neutrophilia with marked leucocytosis (>20 x
109/L)
• Its characterized by presence of immature cells (blasts,
promyelocytes ,myelocytes in peripheral blood(left shift)
• Should be distinguished from Chronic Granulocytic Leukemia (large
number of myelocytes and presence of Philadelphia chromosome
• While Leukamoid reaction has toxic granulation,Dohle bodies and
high NAP score
• Causes include
» Infections
» Marrow infiltration
» Systemic disease ( Acute Glomerulonephritis & Acute liver
failure)
Quantitative changes (NEUTROPENIA) contd.
• Neutropenia is an absolute reduction in the

number of circulating neutrophils
» Mild (1- 1.5 x 109/L)
» Moderate (0.5 – 1 x 109/L)
» Severe (<0.5 x 109/L)
• Symptoms are rare with the neutrophil count above 1 x
109/L
• Bacterial infections are the commonest presentation
• Fungal, viral and parasitic infection are relatively
uncommon
• Causes of Neutropenia
– Racial
– Congenital
– Cyclical neutropenia
– Marrow aplasia
– Marrow infiltration
– Megaloblastic anemia
– Acute infections
• Typhoid, Miliary TB, viral hepatitis
– Drugs
– Irradiation exposure
– Immune disorders
• HIV
• SLE
• Felty’s syndrome
• Neonatal isoimmune and autoimmune neutropenia
– Hypersplenism
• Management of Neutropenia
– Remove the cause if possible
– Treat any infection aggressively
– Role of
– Growth factors
– Splenectomy
• Cyclical neutropenia
– Regular recurring episodes of severe neutropenia (<0.2 x
109/L) usually lasting for 3-6 days
– Can be familial & inherited with maturation arrest
– Three suggested mechanisms for cyclical neutropenia
» Stem cell defect & altered response to growth factors
» Defect in humoral or cellular stem cell control
» Periodic accumulation of an inhibitor
Quantitative changes (LYMPHOCYTOSIS)
• The blood contain only few percent of total body

lymphocytes 1.5-4.0(20-45%)
• The most consistent variation is seen with age
• Alteration of lymphocyte counts can result from
– The redistribution of lymphocytes
» Results in variation in count in serial measurements
– Absolute increase of lymphocyte number
– Loss of lymphocytes
– Combination of these
• Non-malignant causes of lymphocytosis

– Infections
• Viral infections
» Infectious mononucleosis
» CMV
» Rubella, hepatitis, adenoviruses, chicken pox,dengue
• Bacterial infections
» Pertussis
» Healing TB, typhoid fever
• Protozoal infections
» Toxoplasmosis
– Allergic drug reactions
– Hyperthyroidism
– Splenectomy
– Serum sickness
• Infectious Mononucleosis
– Epstein-Barr virus
– Saliva from infected person is the main contagion
– Virus infect epithelial cells and B cells
– Autocrine growth stimulation
– Infection in children under the age of 10 does not cause
illness and result in life long immunity
– Clinical features
– Fever, malaise, fatigue, sore throat, diagnostic red spots at the
junction of soft and hard palate, splenomegaly
– Blood picture shows leucocytosis ( 10 – 20 x 109/L) due to absolute
increase in the number of lymphocytes
– Diagnosis is by serological tests
– There is no specific treatment
Lymphocytosis
Lymphopenia
• Occurs commonly in immunodeficiency
disorders;
• Primary/Congenital
-X-linked hypogammaglobulinemia
-Thymic hyperplasia(Di-George’s syndrome)
-Severe combined immunodeficiency disease
Lymphopenia
• Acquired-HIV,AIDS
-HIV is a retrovirus which infects T-
helper(CD4+)cells
-The virus remains latent in the T-cells, when the
cells are reactivated, the virus replicates and
death follows
Quantitative changes (MONOCYTOSIS)
• Absolute monocyte count is age dependent

• Count in adults 0.2-0.8x 109/L(2-10%)
• Have no marrow reserves
• Causes of monocytosis can be grouped as
– Infections
• Chronic infection (TB, typhoid fever, infective endocarditis)
• Recovery from acute infection
– Malignant disease
• MDS, AML, HD, NHL
– Connective tissue disorders
• Ulcerative colitis, Sarcoidosis, Crohn’s disease
– Post splenectomy
Eosinophilia
• Reactive eosinophilia is common in a wide
variety of allergic and parasitic infections
• Normal count 0.04-0.4(1-6%)
• Eosinophils may be cytologically normal or
show a greater or lesser degree of
degranulation or vacuolation
Quantitative changes (EOSINOPHILIA)
• The causes of eosinophilia can be considered under

following headings
– Allergy
» Atopic, drug sensitivity and pulmonary eosinophilia
– Infection
» Parasites, recovery from infections
– Malignancy
» Hodgkin’s disease, NHL and myeloproliferative disorders
– Drugs
– Skin disorders
– Gastrointestinal disorders
– Hypereosinophilic syndrome
Eosinophilia
• Initial step in investigating unexplained
hypereosinophilia is a full history including a
travel and drug history followed by a physical
examination.
• The lymphocyte count is usually normal and, if
it is elevated, eosinophilia secondary to T-cell
non-Hodgkin’s lymphoma should be
suspected.
Idiopathic Hyperesinophilc
syndrome
• It’s a heterogeneous group of conditions
characterized by persistent unexplained
eosinophilia and tissue damage (e.g. involving
the heart and nervous system)
• Its attributable to the release of eosinophil
granule contents.
• Common in males.
Quantitative changes (EOSINOPHILIA) Contd.
• Hypereosinophilic syndrome
– Criteria of diagnosis
– Peripheral blood eosinophil >1.5 x 109/L
– Persistence of counts more than 6 months
– End organ damage
– Absence of any obvious cause for eosinophilia
– Organ most commonly involved
– Heart
– Lung
– Skin
Quantitative changes (BASOPHILIA)
• Basophils are least common of the granulocytes

• Reference range for adult is 0-01 – 0.1 x
109/L(<1%)
• Most commonly associated with hypersensitivity
reactions to drugs or food
• Inflammatory conditions e.g RA, ulcerative colitis
are also sometime associated with basophilia
• Myeloproliferative disorders
• Chronic myeloid leukemia
Qualitative changes (MORPHOLOGY)
• Congenital
– Pelger-Huet anomaly
– Autosomal dominant
inheritance
– Pseudo-pelger cells
occur in AML and
myelodysplastic
syndromes
– Bilobed and occasional
unsegmented
neutrophils
May-Hegglin anomaly
– Rare,domiunant
inheritance pattern
• Neutrophils contain
basophilic inclusions of
RNA
• Occasionally there is
associated leucopenia
• Thrombocytopenia and
giant platelet are
frequent
Qualitative changes (MORPHOLOGY) contd.
– Alder’s anomaly
• Granulocytes, monocytes and lymphocytes contain
granules which stain purple with Romanowsky stain
• Granules contain mucopolysaccharides
Qualitative changes (MORPHOLOGY) contd.
– Chediak-Higashi syndrome
• Autosomal recessive disorder
• Clinical examibation reveals partial albinism and
hepatosplenomegally
• Have reccurent pyogenic infections
• Giant granules in granulocytes, monocytes and lymphocytes
• Have neutropenia and thrombocytopenia
• Majority die in childhood from infection and hemmorhage
• Treatment is bone marrow transplant
Chediak Higashi Syndrome
Leukemia
Lotodo T.L.C
Department of hematology and
Blood transfusion
Introduction
• These are malignant diseases of the bone marrow
• Broadly classified into:
i)Acute leukemia
-Acute lymphoblastic leukemia
-Acute myeloblastic leukemia
ii)Chronic leukemia
-Chronic lymphocytic leukemia
-Chronic myeloid leukemia(myeloproliferative
disorders)
ACUTE LYMPHOBLASTIC LEUKEMIA
Introduction
• Its a malignant (clonal) disease of the bone
marrow in which early lymphoid precursors
(lymphoblasts)proliferate and replace the
normal hematopoietic cells of the marrow
• Results in a marked decrease in the production
of normal blood cells. Consequently, anemia,
thrombocytopenia and neutropenia
• Lymphoblasts also proliferate in organs other
than the marrow, particularly the liver, spleen,
and lymph nodes.
Clinical…
• Fever - ALL often have fever without any other
evidence of infection
• Increased risk of infection- Infections are common
when the absolute neutrophil count is less than 500/µl
• Anemia – present with fatigue, dizziness, palpitations,
and dyspnea upon even mild exertiion
• Bleeding can be the result of thrombocytopenia due to
marrow replacement
• Disseminated intravascular coagulation (DIC) occur in
10% of pts with ALL
Clinical…
• Lymphadenopathy
• Those with T-cell ALL, present with symptoms
related to a large mediastinal mass, such as
shortness of breath.
• Infiltration of the marrow by massive numbers of
leukemic cells frequently manifests as bone pain
• About 10-20% of ALL patients may present with
left upper quadrant fullness and early satiety due
to splenomegaly
Lab
• CBC- count with differential demonstrates
anemia and thrombocytopenia,may have
high, normal, or low white blood cell (WBC)
count, but they usually exhibit neutropenia
• PBF- confirms the findings of the CBC count.
Circulating blasts are usually seen,
Schistocytes are sometimes seen if DIC is
present.
Lab…
• Bone marrow aspiration and biopsy are the
definitive diagnostic tests to confirm the
diagnosis of leukemia
• Aspiration slides should be stained for
morphology with leishmania or Giemsa stains
• The diagnosis of acute lymphoblastic
leukemia (ALL) is made when at least 20%
lymphoblasts (WHO) are present in the bone
marrow and/or peripheral blood.
Lab…
• L1 – Small cells with homogeneous chromatin,

regular nuclear shape, small or absent nucleolus,
and scanty cytoplasm
• L2 – Large and heterogeneous cells,
heterogeneous chromatin, irregular nuclear
shape, and nucleolus often large
• L3 – Large and homogeneous cells with multiple
nucleoli, moderate deep blue cytoplasm, and
cytoplasmic vacuolization that often overlies the
nucleus (most prominent feature)
Lab
Lab…
• Cytochemical stains- to differentiate ALL from
AML
• Immunophenotyping-done by Flow
cytometry-CD19,CD10,CD3
• Cytogenetic studies-abnormalities of
chromosome number (hypodiploidy,
hyperdiploidy) are more common in ALL than
in AML.
Commonly employed stains in leukemia diagnosis
Stain Comments
Sudan black B Myelomonocytic cells
Myeoperoxidase Myelomonocytic cells
Chloroacetate esterase Granulocyte blasts
Alpha naphtybutyrate esterase Monocytic cells
Periodic acid- Schift Lymphoblast, erythroblasts -

block positivity
Myeloblast-speckle pattern
Lab…
Biochemical tests
• Elevated lactate dehydrogenase (LDH) level
• Elevated uric acid level
• Elevated serum calcium and magnesium
• Deranged Liver function tests
• ElevatedBlood urea nitrogen (BUN)/creatinine
level
Principles of management
i)Supportive treatment.
a)Maintain Hb>10g/dl by transfusion of packed cells.
b)Platelet transfusions are indicated when:
-There is clinical evidence of bleeding e.g. purpura
-Prophylactic platelet transfusions are often used when
the count fall below
20 x 109/L.
Principles…
-Prevent uric acid nephropathy and gout due to rapid cell
lysis:
-Start allopurinol 300 daily preferably at least 24 hours
before chemotherapy.
-Ensure good fluid input and output with careful records
of balance.
- Check electrolytes daily or more frequently during initial
stages of treatment cell lysis may cause hyperkalaemia
-Antibiotics for infections
-Palliative care
Principle….
• Definitive-There are 4 components of ALL
treatment: -
i)Induction- chemotherapy is given over the
course of 4-6 weeks. Complete remissions
(CRs) are obtained in 65-85% of patients
ii)Consolidation
iii)Maintenance
iv)Central nervous system (CNS) prophylaxis
ACUTE MYELOID LEUKEMIA
AML
• Malignant disease of the bone marrow in
which hematopoietic precursors are arrested
in an early stage of development.
• Distinguished from other related blood
disorders by the presence of more than 20%
blasts in the bone marrow(WHO)
• Incidence in the United states is 2.1/100,000
persons per year
FAB Classification
• M0 - Undifferentiated leukemia
• M1 - Myeloblastic without differentiation
• M2 - Myeloblastic with differentiation
• M3 - Promyelocytic
• M4 – Myelomonocytic; M4eo - Myelomonocytic with
eosinophilia
• M5 - Monoblastic leukemia; M5a - Monoblastic without
differentiation; M5b - Monocytic with differentiation
• M6 - Erythroleukemia
• M7 - Megakaryoblastic leukemia
M1 M2 M3
M5 M6 M7
M3 – Acute Promyelocytic Leukemia
• Bone marrow is almost

completely replaced by
promyelocytes with
abundant cytoplasm
containining
azurophilic granules
and multiple auer rods
WHO Classification
i)AML with recurrent genetic abnormalities - AML with t(8;21)(q22;q22),
(AML1/ETO); AML with abnormal bone marrow eosinophils and
inv(16)(p13q22) or t(16;16)(p13)(q22), (CBFB/MYH11); APL with
t(15;17)(q22;q12), (PML/RARa) and variants; AML with 11q23 (MLL)
abnormalities
ii)AML with multilineage dysplasia - Following myelodysplastic syndrome
(MDS) or MDS/myeloproliferative disease (MPD); without antecedent
MDS or MDS/MPD but with dysplasia in at least 50% of cells in 2 or more
lineages
iii)AML and MDS, therapy related - Alkylating agent or radiation-related type;
topoisomerase II inhibitor type; others
iv)AML, not otherwise classified - AML, minimally differentiated; AML,
without maturation; AML, with maturation; acute myelomonocytic
leukemia; acute monoblastic or monocytic leukemia; acute erythroid
leukemia; acute megakaryoblastic leukemia; acute basophilic leukemia;
acute panmyelosis and myelofibrosis; myeloid sarcoma
Clinical..
• Etiology-genetic(bloom syndrome,Fanconi’s
anemia),enviromental(benzene,radiation
alkylating agents)
• Symptoms results from bone marrow failure,
organ infiltration with leukemic cells, or both
• Patients with APL(M3) present commonly with
DIC
• Sternal tenderness and gingival hyperplasia may
be presenting sign in monocytic leukemias
CHRONIC LYMPHOCYTIC LEUKEMIA
CLL
• Its a monoclonal disorder characterized by a
progressive accumulation of functionally
incompetent lymphocytes
• Most common form of leukemia found in adults
• Onset is insidious, and it is not unusual for CLL to
be discovered incidentally after a blood cell count
is performed for another reason
• Enlarged lymph nodes are the most common
presenting symptom
Lab…
• CBC - shows absolute lymphocytosis, with
more than 5000 B-lymphocytes/µL.
• Lymphocytosis must persist for longer than 3
months
• Bone marrow aspiration, biopsy and flow
cytometry may be necessary to assess other
complicating features such as anemia and
thrombocytopenia.
Lab
• PBF-confirm
lymphocytosis. It
usually shows the
presence of smudge
cells
• Lymphnode biopsy may
be taken for histology
Staging
• Two staging systems are in common use for
CLL: the modified Rai staging in the United
States and the Binet staging in Europe
• The revised Rai staging system divides patients
into low-, intermediate-, and high-risk groups
Rai staging
• Low risk – Lymphocytosis in the blood and
marrow only
• Intermediate risk – Lymphocytosis with
enlarged nodes in any site or splenomegaly or
hepatomegaly
• High risk – Lymphocytosis with disease-
related anemia (hemoglobin < 11 g/dL) or
thrombocytopenia (platelets < 100 x 109/L
Binet Staging
• Stage A – Hemoglobin greater than or equal to
10 g/dL, platelets greater than or equal to 100
× 109/L, and fewer than 3 lymph node areas
involved.
• Stage B – Hemoglobin and platelet levels as in
stage A and 3 or more lymph node areas
involved
• Stage C – Hemoglobin less than 10 g/dL or
platelets less than 100 × 109/L, or both
Principles of ma…
• Chemotherapy is not needed in CLL until
patients become symptomatic or display
evidence of rapid progression of disease
• A variety of chemotherapy regimens are used
in CLL they include: nucleoside analogues and
alkylating agents often in combination
• Allogeneic stem cell transplantation is the
only known curative therapy
END
• Questions?
CHRONIC MYELOGENOUS
LEUKEMIA
CML
• Characterized by increased proliferation of
the granulocytic cell line without the loss of
their capacity to differentiate
• Consequently, the peripheral blood cell
profile shows an increased number of
granulocytes and their immature precursors,
including occasional blast cells.
• The tyrosine kinase activity of the bcr-abl
hybrid gene is increased.
CML
• CML is one of the few cancers known to be
caused by a single, specific genetic mutation.
• More than 90% of cases result from a cytogenetic
aberration known as the Philadelphia
chromosome
• CML progresses through 3 phases: chronic,
accelerated, and blast
• In the chronic phase of disease, mature cells
proliferate; in the accelerated phase, additional
cytogenetic abnormalities occur; in the blast
phase, immature cells rapidly proliferate
CML
• Approximately 85% of patients are diagnosed in
the chronic phase and then progress to the
accelerated and blast phases after 3-5 years
• CML accounts for 20% of all leukemias affecting
adults
• It typically affects middle-aged individuals.
Uncommonly, the disease occurs in younger
individuals
• Younger patients may present with a more
aggressive form of CML, such as in accelerated
phase or blast crisis
CML
• It is characterized by a
cytogenetic aberration
consisting of a
reciprocal translocation
between the long arms
of chromosomes 22
and 9 t(9;22)
• The translocation
results in a shortened
chromosome 22
CML
• This translocation relocates an oncogene called ABL
from the long arm of chromosome 9 to a specific
breakpoint cluster region (BCR) in the long arm of
chromosome 22
• The resulting BCR/ABL fusion gene encodes a chimeric
protein with strong tyrosine kinase activity.
• The presence of BCR/ABL rearrangement is the
hallmark of CML, although this rearrangement has also
been described in other diseases
• It is considered diagnostic when present in a patient
with clinical manifestations of CML.
Lab
• CBC- the increase in mature granulocytes and
normal lymphocyte counts, results in a total
WBC count of 20,000-60,000 cells/Μl
• A mild increase in basophils and eosinophils
is present and becomes more prominent
during the transition to acute leukemia.
• The diagnosis of CML is based on the findings
in the peripheral blood and the Philadelphia
(Ph1) chromosome in bone marrow cells
Lab…
Principles of mx
• The goals of treatment of CML are threefold:
i)Hematologic remission (normal complete
blood cell count [CBC] and physical
examination
ii)Cytogenetic remission (normal chromosome
returns with 0% Ph-positive cells)
iii)Molecular remission (negative polymerase
chain reaction [PCR] result for the mutational
BCR/ABL gene
Principles….
• The chronic phase varies in duration,
depending on the maintenance therapy used:
it usually lasts 2-3 years with hydroxyurea
(Hydrea) or busulfan therapy
• Blast phase is managed as AML
Lymphoma
Lotodo T.L.C
Department of Hematology and
Blood Transfusion
Objectives
• Classification of lymphomas
• Etiology and pathogenesis of lymphomas
• Clinical presentation
• Laboratory evaluation
• Principles of Management
Introduction
• Neoplastic (clonal) lymphoproliferative disorder
primarily involving solid lymphoid organs.
• Lymphoid cells may transform into malignant cells at
any time during maturation, giving rise to a
malignancy that is specific to the stage in which the
cell becomes transformed.
• Malignancies of lymphoid cells almost always present
as Leukemia (widespread involvement of bone
marrow accompanied by the presence of large
numbers of tumor cells in the peripheral blood),as
solid tumors (lymphoma) or both lymphoma and
leukemia
Etiology and pathogenesis
 Viral
(i)EBV-Burkitts
 -Post organ transplant lymphoma
 -Primary CNS diffuse large B-cell lymphoma
 -Hodgkins disease
 - Extranodal NK/Tcell lymphoma,nasal type
(ii)HTLV I-Adult T-cell lymphoma
(iii)HIV-Diffuse large B-cell lymphoma and Burkitts,
plasma cell myeloma
Etiology…
• Hepatitis C-lymphoplasmacytic lymphoma,CLL
• H Pylori-Gastric MALT lymphoma due to
chronic immune reaction to bacterium
• HHV8-Primary effusion lymphoma
• Other etiological factors are inherited and
acquired immunodeficiencies,autoimmune
diseases, chemicals and radiation
Classification….
• Two major categories

– Non-Hodgkin’s lymphoma(~70%)
– Hodgkin’s lymphoma (~30%)
• Both are distinct in:
– Morphology
– Immunophenotype
– Clinical features and treatment
HODGKIN’S LYMPHOMA
Introduction
• Thomas Hodgkin first described the disorder
in 1832
• Have a bimodal distribution in both sexes,
peaking in young adults (aged 15-34 y) and
older individuals (>55 y)
HL
• In classic Hodgkin
lymphoma, the neoplastic
cell is the Reed-Sternberg
(RS) cell
• Reed-Sternberg cells
comprise only 1-2% of the
total tumor cell mass.
• The remainder is composed
of a variety of reactive,
mixed inflammatory cells
consisting of lymphocytes,
plasma cells, neutrophils,
eosinophils, and histiocytes
Classifi……WHO
• Hodgkin lymphoma exists in 5 types
• Four of these:
i)Classic
-nodular sclerosis
- mixed cellularity
- lymphocyte depleted
-lymphocyte rich
ii) Nodular lymphocyte predominant Hodgkin
disease (NLPHD)
Nodular Sclerosis Hodgkin Disease
• Constitutes 60-80% of all cases of Hodgkin
lymphoma
• Morphology shows a nodular pattern. Broad
bands of fibrosis divide the node into nodules.
• The capsule is thickened
• The characteristic cell is the lacunar-type Reed-
Sternberg cell, which has a monolobated or
multilobated nucleus, a small nucleolus, and
abundant pale cytoplasm.
Mixed-Cellularity Hodgkins Disease
• Constitutes 15-30% of cases, the infiltrate is
usually diffuse.
• Reed-Sternberg cells are of the classic type
(large, with bilobate, double or multiple
nuclei, and a large, eosinophilic nucleolus)
• Commonly affects the abdominal lymph nodes
and spleen
• Commonly observed in patients with human
immunodeficiency virus infection.
Lymphocyte-rich Hodgkins Disease
• Constitutes 5% of cases
• Reed-Sternberg cells of
the classic or lacunar
type are observed, with a
background infiltrate of
lymphocytes
• Some cases may have a
nodular pattern.
• Clinically, the
presentation and survival
patterns are similar to
those for MCHD.
Lymphocyte-depleted Hodgkins
disease
• Constitutes less than 1% of cases
• The infiltrate in LDHD is diffuse and often
appears hypocellular.
• Large numbers of Reed-Sternberg cells and
bizarre sarcomatous variants are present.
• LDHD is associated with older age and HIV-
positive status
• Epstein-Barr virus (EBV) proteins are expressed in
many of these tumors
Nodular lymphocyte-predominant
Hodgkins disease
• Constitutes 5% of cases
• In contrast to the other histologic subtypes, the typical
Reed-Sternberg cells are either infrequent or absent
in NLPHD
• Lymphocytic and histiocytic (L&H) cells, or "popcorn
cells" (their nuclei resemble an exploded kernel of
corn), are seen within a background of inflammatory
cells, which are predominantly benign lymphocytes.
• Unlike Reed-Sternberg cells, L&H cells are positive for
B-cell antigens, such as CD19 and CD20, and are
negative for CD15 and CD30.
Clinical findings..hist
• Asymptomatic lymphadenopathy
• Constitutional symptoms (eg, unexplained weight loss,
fever, night sweats) known as "B symptoms."
• Intermittent fever, the classic Pel-Ebstein fever is observed
(high fever for 1-2 wk followed by an afebrile period of 1-2
wk)
• Chest pain, cough, shortness of breath, or a combination of
these things may be present due to a large mediastinal
mass or lung involvement; rarely, hemoptysis occurs
• Patients may present with pruritus
• Pain at sites of nodal disease, precipitated by drinking
alcohol, is specific for Hodgkin lymphoma
• Back or bone pain may rarely occur
Clinical…physical exam
• Palpable, painless lymphadenopathy can be seen in
the cervical area (neck, 60-80%), axilla (armpit, 6-
20%), and, less commonly, in the inguinal area (groin,
6-20%); it is described as rubbery adenopathy
• Splenomegaly and/or hepatomegaly may be present
• Superior vena cava syndrome may develop in patients
with massive mediastinal lymphadenopathy
• Central nervous system symptoms or signs may be due
to paraneoplastic syndromes, including cerebellar
degeneration, neuropathy, Guillain-Barre syndrome, or
multifocal leukoencephalopathy
Lab…
• CBC count studies for anemia, lymphopenia
neutrophilia or eosinophilia
• ESR- The erythrocyte sedimentation rate may be
elevated in Hodgkin lymphoma.
• LDH-Lactate dehydrogenase may be increased
• Serum creatinine may be elevated, in the rare
cases of nephrotic syndrome associated with
Hodgkin lymphoma
• ALP may be increased due to the presence of
liver or bone involvement
Lab…
• Biopsy-a histologic diagnosis of Hodgkin
lymphoma is always required
• Bone marrow aspirates and biopsies -bone
marrow involvement is more common in patients
who are elderly or have advanced-stage disease,
systemic symptoms, or a high-risk histology.
• Because Hodgkin lymphoma in the bone marrow
is often patchy, bilateral bone marrow biopsies
are advised to improve yield
Lab…
• Pleural effusion-Sampling of a pleural effusion
by thoracocentesis and examination of the
cells obtained may be useful in the evaluation
of Hodgkin lymphoma
• CSF- CNS evaluation by lumbar puncture
Staging
• The Ann Arbor classification (1971) is used most often
for Hodgkin lymphoma
• It classifies cases into the following 4 stages,
principally on the basis of lymph node involvement:
• Stage I - a single lymph node area or single extranodal
site
• Stage II - 2 or more lymph node areas on the same side
of the diaphragm
• Stage III - denotes lymph node areas on both sides of
the diaphragm
• Stage IV - disseminated or multiple involvement of the
extranodal organs
Staging
• A or B designations denote the absence or
presence of B symptoms
• "B" designation includes the presence of 1 or
more of the following:
-Fever (temperature >38°C)
-Drenching night sweats
-Unexplained loss of more than 10% of body
weight within the preceding 6 months
Principles of mana….
• Treatment of Hodgkin lymphoma is tailored to
disease type, disease stage, and an assessment of
the risk of resistant disease.
• Hodgkin lymphoma is considered to be a curable
malignancy, but therapies for this disease can
have significant long-term toxicity
• Combined-modality therapy (radiation therapy
[XRT] and chemotherapy) is frequently the
preferred approach in for most patients
NON HODGKINS LYMPHOMA
Intro…
• About 85% of all malignant lymphomas are NHLs
• The median age at diagnosis is the sixth decade of life,
although Burkitt lymphoma and lymphoblastic
lymphoma occur in younger patients
• Almost 85% of NHLs are of B-cell origin; only 15% are
derived from T/NK cells, and the small remainder stem
from macrophages
• These tumors are characterized by the level of
differentiation, the size of the cell of origin, the origin
cell's rate of proliferation, and the histologic pattern of
growth.
Classification
• The Working Formulation, originally proposed in 1982
classified lymphomas into low,intermediate, and high
grade
• In the 1990s, the Revised European-American
Lymphoma (REAL) classification attempted to apply
immunophenotypic and genetic features in identifying
distinct clinicopathologic NHL entities
• The World Health Organization (WHO) classification
further elaborates upon the REAL approach
• It divides NHL into those of B-cell origin and those of
T-cell and natural killer (NK)–cell origin.
B cell neoplasms
• Precusor Bcell neoplasms
• Precusor B lymphoblastic leukemia/lymphoma
• Mature B cell neoplasms
• 1.CLL/SLL
• 2.Bcell prolymhocytic leukemia
• 3.Lymphoplasmacytic lymphoma
• 4.Splenic marginal cell lymphoma
• 5.Hairy cell leukemia
• 6.Diffuse large Bcell lymhoma(mediastinal B
cell, plasmablastic Tcell, ALK-positive,
intravascular,primary effusion)
CONT
• 7.Mantle cell lymphoma
• 8.Plasma cell myeloma
• 9.Follicular lymphoma
• 10.Solitary plasmacytoma of bone
• 11Extaosseous plasmacytoma
• 12Extranodal marginal zone Bcell
lymphoma(MALT-lymphoma
• 13Nodal marginal zone lymphoma
• 17Burkitt lymphoma leukemia
T-cell and Nk-cell neoplasms
 Precusor Tcell neoplasms
 1.Precusor T lymphoblastic leukemia/lymphoma
 2.Blastic Nk cell lymphoma
 Mature Tcell and NK cell neoplasms
 1.Tcell prolymhocytic leukemia
 2.Tcell large granular lymhocytic leukemia
 3.Aggresive NK cell leukemia
 4.Adult Tcell leukemia
 5.Etranodal NK/Tcell lymphoma,nasal type
 6.Eteropathy type Tcell lymphoma
Classi….
 7.Hepatosplenic Tcell lymphoma
 8.Subcuteneous panniculitis like Tcell lymphoma
 9.Mycosis fungoides
 10.Sezzary syndrome
 11.Angioimmunoblastic Tcell lymphoma
 12.Anaplastic large cell lymphoma
 13.Primary cutenous anaplastic large cell
lymphoma
 14.Peripheral large cell lymphoma ,unspecified.
Burkitt Lymphoma
• Its a highly aggressive B-cell non-Hodgkin
lymphomas that is characterized by the
translocation and deregulation of the c-myc
gene on chromosome 8
• EBV was found to play a role in pathogenesis of
BL by de-regulation of the oncogene c-MYC
• All endemic tumors are infected with EBV
• It’s the commonest lymphoma in HIV patients
(25%)
• Its characterized as being poorly responsive to
CHOP
BL
• Burkitt lymphoma (BL) is named after Dr. Dennis
Burkitt, an Irish surgeon who worked in Kampala,
Uganda, and noted a large number of African
children affected with rapidly growing jaw
tumors in falciparum malaria endemic areas.
• A few years later, Dr. Burkitt met and provided
tumor samples to Dr. Epstein, who was a
pathologist. Dr. Epstein and his colleagues
identified Epstein-Barr virus from the lymphoma
samples, which established a role for EBV in the
pathogenesis of African Burkitt lymphoma
BL
• Its one of the fastest
growing malignancies in
humans, with a growth
fraction close to 100%
and a doubling time of
around 25 hours
• Commonly presenting in
children and young
adults, with frequent
bone marrow and central
nervous system (CNS)
involvement
BL
• Characterized by a
monoclonal proliferation of
medium-sized, noncleaved
B-cells that are uniform in
appearance and that
produce a diffuse pattern of
tissue involvement
• These cells typically have a
scant basophilic cytoplasm
characterized by numerous
lipid vacuoles, round nuclei
with stippled chromatin and
multiple small nucleoli.
BL
• Hallmark of Burkitt
lymphoma (BL) is the
presence of a "starry
sky" appearance
imparted by scattered
macrophages
phagocytizing cell
debris and apoptotic
cells (tingible body
macrophages)
Multiple myeloma
Lotodo T.LC
Blood Transfusion
Intro…
• Its a debilitating malignancy that is part of a
spectrum of diseases ranging from monoclonal
gammopathy of unknown significance(MGUS) to
plasma cell leukemia
• MM is characterized by a proliferation of malignant
plasma cells and a subsequent overabundance of
monoclonal paraprotein (M protein)
Intro…
• The cells may cause
soft-tissue masses
(plasmacytomas) or
lytic lesions in the
skeleton.
• Complications of MM
are bone pain,
hypercalcemia, renal
failure, and spinal cord
compression.
Poor prognostic factors
• Tumor mass
• Hypercalcemia
• Bence Jones proteinemia
• Renal impairment (ie, stage B disease or
creatinine level >2 mg/dL at diagnosis)
Investigations
• Serum and urine assessment for monoclonal
proteins
• Bone marrow aspiration and/or biopsy
• Serum beta(2)-microglobulin, albumin, and
lactate dehydrogenase measurement
• Skeletal survey
• MRI
Lab..
• CBC- to determine if the patient has anemia,
thrombocytopenia, or leukopenia abnormal
coagulation, and an increased ESR
• The reticulocyte count is typically low
• Peripheral blood smears may show Rouleaux
formation.
Lab…
• Metabolic panel to assess levels of total
protein, albumin and globulin, blood urea
nitrogen (BUN), creatinine, and uric acid
• A 24-hour urine - for quantification of the
Bence Jones protein (ie, lambda light chains),
protein, and creatinine clearance.
Lab…
• Quantification of proteinuria is useful for the
diagnosis of MM (>1 g of protein in 24 h is a
major criterion) and for monitoring the
response to therapy
• Creatinine clearance can be useful for
defining the severity of the patient’s renal
impairment.
Lab…
• Serum protein electrophoresis (SPEP) is used
to determine the type of each protein present
and may indicate a characteristic curve (ie,
where the spike is observed)
• Immunofixation is used to identify the
subtype of protein (ie, IgA lambda).
Lab…
Lab…
• BMA and Biopsy- show
sheets or clusters of
plasma cells
• Biopsy enables a more
accurate evaluation of
malignancies than does
bone marrow
aspiration.
Lab..
• Plasma cells are 2-3
times larger than typical
lymphocytes; they have
eccentric nuclei that
are smooth,with
clumped chromatin
and have a perinuclear
halo The cytoplasm is
basophilic.
Radio…
• Plain radiography can
usually depict lytic
lesions
• Such lesions appear as
multiple, rounded,
punched-out areas
found in the skull,
vertebral column, ribs,
and/or pelvis
Principles of mx…
• Care of patients with MM is complex and
should focus on treatment of the disease
process and any associated complications
• Although MM remains incurable, several drug
therapies are valuable in the treatment of
patients with MM, as are autologous stem cell
transplantation, radiation, and surgical care
in certain cases.
APLASTIC ANAEMIA
Dr Kipkulei
Definition
• Syndrome of bone marrow failure
characterized by peripheral pancytopenia and
bone marrow hypoplasia resulting from failure
or suppression of multipotent myeloid stem
cells
Aetiology
a. Congenital- 20% of cases
• Fanconi anemia
• Dyskeratosis congenita
b. Acquired- 80% of causes
i. Idiopathic- 65% of acquired causes
ii. Secondary causes
• Radiation
• Chemicals - benzene, glue vapours
Aetiology(cont.)
ii. Acquired secondary causes(cont.)
• Drugs- chloramphenicol, gold, cytotoxics
• Viruses- EBV, hepatitis(non-A, non-B. non-C),
HIV, parvovirus
• Immune diseases- eosinophilic fascitis, SLE,
GvHD
• Paroxysmal Nocturnal Hemoglobinuria
• Pregnancy
Pathophysiology
• Not fully understood
• Two major mechanisms proposed:
Immunologically mediated suppression- ↑
activated cytotoxic T lymhocytes→ ↑gamma
interferon, ↑TNF, ↑IL-2; ↑HLA-DR2
Intrinsic abnormality of stem cells
Epidemilogy
 Global incidence- 2-4 cases/year in the USA
and Europe; 6-14 cases/year in Asia
 Local statistics- ??
 Sex- male:female 1:1
 Age- occurs in all age groups; peak incidence
at age 20-25 years and >60years
 Mortality/morbidity- major causes include
infection and bleeding
Clinical features
Symptoms
• Onset is often insidious, but may be abrupt
• Symptoms are related to the cytopenias:
– Anemia- symptoms of anemia
– Thrombocytopenia- bleeding tendencies
– Leocopenia/neutropenia- bacterial and fungal
infections
Clinical features(cont.)
Signs
• Signs of anemia
• Signs of thrombocytopenia- petechiae,
purpura or ecchymoses
• Signs of infections- e.g. fever, though overt
signs are usually not apparent at presentation
• Look for signs of physical stigmata of
congenital marrow failure syndromes
Diagnosis
• History
• Physical examination
• Laboratory invetigations:
i. Complete blood count- pancytopenia
ii. Peripheral blood smears- important in
distinguishing aplasia from infiltrative and
myelodyplastic syndomes
Diagnosis(cont.)
• Laboratory investigations(cont.)
iii. Bone marrow aspiration and biopsy:
 Hypocellular marrow- largely empty marrow
spaes, fat cells, fibrous stroma, scattered
foci of lymphocytes/plasma cells
 Dyserythropoiesis and megaloblasosis may
be observed
Diagnosis(cont.)
• Laboratory investigatiosns(cont.)
iv. Hb electrophoresis- may show elevated fetal
Hb suggesting stress erythropoiesis
v. Tests to evaluate etiology and differential
diagnosis- Coomb’s test, U/E/C, liver
function tests, viral serological tests,
radiological tests etc
Classification of aplastic anemia
 Classification is based on the criteria of the
International Aplastic Anemia Study Group
 Criteria is based on peripheral blood and BM
findings
 Peripheral blood:
 absolute reticulocyte count <40,000(<1%)
Classification(cont.)
• Peripheral blood(cont.)
Absolute neutrophil count(ANC) <0.5x109/L
Platelet count <20x109/L
 Bone marrow:
Marrow of <30% normal cellularity
Classification(cont.)
a. Severe Aplastic Anemia(SAA)-
 2 of 3 abnormal peripheral blood values in
addition to the bone marrow change
b. Very Severe Aplastic Anemia(VSAA)
 Features of SAA with ANC <0.2x109/L
Differential diagnosis (other causes of
pancytopenia)
• ALL
• AML
• Myelodysplastic syndromes
• Hyersplenism
• BM replacement with tumor or fibrosis
• Severe megaloblastic anemia
• Overwhelming infection- HIV, EBV
• PNH
Treatment
• Supportive therapy
• Remove offending agents
• Selective transfusion therapy to avoid
sensitization
• Treatment of infections- broad spectrum
antibiotics and antifungal agents
Treatment(cont.)
Definitive therapy
a) Immunosupressive therapy
• NOT curative
• Goal is sustained remission
 20-30% will have recurrent aplastic anemia
 20-30% develop clonal disorder: MDS or
acute leukemia
Treatment(cont.)
Definitive therapy(cont.)
a) Immunosupressive therapy(cont.)
• Combination therapy is best
 Antithymocyte globulin (ATG)
 Cyclosporine
 High dose corticosteroids
e.g.methylprednisolone
NB: patients with VSAA respond poorly to
immunosuppressive therapy
Treatment(cont.)
b) Bone marrow transplantation (BMT)
• Therapy choice influenced by age and
disease severity
i. <20yrs old- Allogeinic BMT with matched
sibling
 50-60% cure rate,
 Condition pretransplant with
ATG/Cyclophosphamide/cyclosporine
Treatment(cont.)
BMT(cont.)
ii. 20-25 yrs old- BMT if in excellent health with
fully matched sibling
iii. >45yrs old
 ?Immusupressive therapy only
 ?BMT with conditioning before BMT
showing increased survival
Treatment(cont.)
• Cytokines may be indicated in cases of VSAA
or failure to respond to immunosupressive
therapy
Recombinant human GM-CSF-
Sargramostin(Leukine, Prokine)
Recombinant G-CSF- Filgrastim(Neupogen)
Prognosis
• Untreated aplastic anemia leads to rapid
death within 6 months
• Outcome has improved with supportive care
• 5 yr survival rate of those receiving
immunosupressive therapy is 75% and those
receiving BMT from a matched sibling is >90%
Reference
1. Postgraduate hematology, A.V. Hoffbrand,
S.M. Lewis
2. eMedicine Hematology:
www.emedicine.medscape.com
3. MedlinePlus Medical Encyclopedia
Myeloproliferative Disorders
Lotodo T.L.C
Blood transfusion
Intro…
• Heterogenous group of disorders
characterized by cellular proliferation of one
or more hematologic cell lines in the bone
marrow and also in other hemopoietic organs
e.g liver and spleen
• Myeloproliferative disease may evolve into
one of the other myeloproliferative
conditions, transform to acute leukemia or
both
Classification of MPDs
FAB WHO
Chronic myelogenous leukemia(CML) Chronic Myelogenous Leukemia
Polycythemia Vera(PV) Polycythemia Vera
Essential Thrombocythemia(ET) Essential Thrombocytosis
Agnogenic myeloid Chronic idiopathic myelofibrosis

metaplasia/myelofibrosis
Chronic eosinophilic
leukemia/hyperesinophilic syndrome
Polycythaemia Vera
• Increase in hemoglobin concentration above
upper limit for age and sex
• The most prominent feature of this disease is
an elevated absolute red blood cell mass
because of uncontrolled red blood cell
production
• Its accompanied by increased white blood
cell (myeloid) and platelet (megakaryocytic)
production
Intro…
• PV can be described as absolute(red cell mass
is raised) or relative(plasma volume is
reduced)
• Absolute PV can be further subdivided into
primary Polycythaemia rubra vera)or
secondary polycythaemia
Etiology
i)Primary- Valine to phenylalanine substitution
at amino acid position 617 (V617F) within the
Janus kinase 2 (JAK2) gene,this produces
hypersensitivity to erythropoietin
ii)Secondary-high altitudes,pulmonary
disease,heart diseases,renal disease
iiiRelative-dehydration,plasma loss in burns
Clinical presentation
• Symptoms are related to hyperviscosity, sludging
of blood flow, and thromboses leads to lead to
headache, dizziness, vertigo, tinnitus, visual
disturbances
• Bleeding complications- epistaxis, gum bleeding,
ecchymoses, and gastrointestinal (GI) bleeding.
• Thrombotic complications- venous thrombosis or
thromboembolism and an increased prevalence
of stroke and other arterial thromboses.
• Abdominal pain due to peptic ulcer
disease,splenomegaly and pruritis
Criteria for diagnosis of PRV
• Category A Category B
-Total red cell mass -Platelets>400x109/l
male>35ml/kg -White cells >12x109/l
Female>32ml/kg -Increased NAP score
-Arterial oxygenation>92% -Raised Vit B12 level
-Splenomegally
Lab…CBC,PBF
• The red blood cells are normochromic normocytic,
unless the patient has been bleeding from underlying
peptic ulcer disease or phlebotomy treatment which
will be hypochromic and microcytic, reflecting low iron
stores
• An elevated white blood cell count (>12,000/µL)
occurs in approximately 60% of patients
• It is mainly composed of neutrophils with a left shift
and a few immature cells
• Mild basophilia occurs in 60% of patients.
• The leukocyte ALP score is elevated (>100 U/L) in 70%
of patients
Lab…CBC,PBF
• The platelet count is elevated to 400,000-
800,000/µL in approximately 50% of patients
• Morphologic abnormalities in platelets include
macrothrombocytes and granule-deficient
platelets.
Lab…BMA
• Bone marrow studies show hypercellularity and
hyperplasia of the erythroid, granulocytic, and
megakaryocytic cell lines or myelofibrosis
• Iron stores are decreased or absent because of
the increased red blood cell mass, and
macrophages may be masked in the myeloid
hyperplasia that is present.
• Fibrosis is increased and detected early by silver
stains for reticulin.
Lab..
• The serum Epo level should be decreased in
nearly all patients with polycythemia vera (PV)
and no recent hemorrhage
• This distinguishes primary polycythemia from
secondary causes of polycythemia in which
the serum Epo level is generally within the
reference range or is elevated
PV-Management
• Phlebotomy or bloodletting has been the
mainstay of therapy
• Once the patient's hemoglobin and
hematocrit values are reduced to within the
reference range (ie, < 45%), implement a
maintenance program either by inducing iron
deficiency by continuous phlebotomies or
using a myelosuppressive agent,hydroxyurea
PV-management
• Hydroxyurea (Droxia, Hydrea) or anagrelide
(Agrylin) -- reduces number of blood cells.
• Low-dose aspirin - reduces skin redness and
burning, and lowers increased temperature
that may occur with the condition.
• Antihistamines - decreases itching.
• Allopurinol - reduces symptoms of gout, a
potential complication of polycythemia
Essential Thrombocytosis
• There is sustained increase in platelet count
because of megakaryocyte proliferation
• Platelet counts are >600x109/l
• Present with thrombosis and hemorrhage
• Also may have burning sensation of hands
and feet
• May have splenomegaly or splenic atrophy
due to infarction
Other causes of thrombocytosis
• Hemorrhage,trauma • PBF- large platelets and
• Chronic iron deficiency megakaryocytes
• Malignancy • BMA- Excess abnormal
• Chronic infections magakaryocyte
• Connective tissue
diseases
• Post splenectomy
Essential Thrombocytosis
ET-management
• Low-dose aspirin -- may treat headache and
burning pain in the skin.
• Hydroxyurea (Droxia, Hydrea) or anagrelide
(Agrylin) -- reduces number of blood cells.
• Aminocaproic acid -- reduces bleeding. This
treatment may be used before surgery to
prevent bleeding as well.
Myelofibrosis
• Progressive generalized fibrosis of bone
marrow in association with development of
hemopoiesis in the spleen and liver
• Clinically leads to anemia and hepato-
splenomegaly
• Fibroblasts are stimulated by platelet derived
growth factor and other proteins secreted by
megakaryocytes and platelets
Lab..CBC,PBF
• Anemia-Causes include hemodilution,
ineffective erythropoiesis, and shortened
RBC survival
• Peripheral blood reveals
leukoerythroblastosis with teardrop
poikilocytosis
• Large platelets and megakaryocyte
fragments may be observed.
Lab..CBC,PBF
• Leukopenia is observed in up to 25% whereas
leukocytosis may be observed in one third.
• A small number of blasts and Pelger-Huet
cells are observed
• Thrombocytosis is more common than
thrombocytopenia
• DIC is observed in 15% of patients.
Lab..Bone marrow Biopsy
• This is usually performed over the posterior
iliac crest, using specialized needles
• Biopsy specimens should not be obtained
from the sternum, sternal aspirates are
typically not useful because and the inability
to obtain a biopsy from this site
• Biopsy specimens reveal hypercellular
marrow with increased megakaryocytes.
Lab…BMA
•
Myelofibrosis-Management
• Hydroxyurea -- may control complications,
such as enlargement of the liver and spleen,
reduce the number of white cells and platelets
in the blood, and improve anemia.
• Thalidomide and lenalidomide - to reduce
symptoms and treat anemia
CML
• Characterized by increased proliferation of
the granulocytic cell line without the loss of
their capacity to differentiate
• Consequently, the peripheral blood cell
profile shows an increased number of
granulocytes and their immature precursors,
including occasional blast cells.
• The tyrosine kinase activity of the bcr-abl
hybrid gene is increased.
CML
• CML is one of the few cancers known to be
caused by a single, specific genetic mutation.
• More than 90% of cases result from a cytogenetic
aberration known as the Philadelphia
chromosome
• CML progresses through 3 phases: chronic,
accelerated, and blast
• In the chronic phase of disease, mature cells
proliferate; in the accelerated phase, additional
cytogenetic abnormalities occur; in the blast
phase, immature cells rapidly proliferate
CML
• Approximately 85% of patients are diagnosed in
the chronic phase and then progress to the
accelerated and blast phases after 3-5 years
• CML accounts for 20% of all leukemias affecting
adults
• It typically affects middle-aged individuals.
Uncommonly, the disease occurs in younger
individuals
• Younger patients may present with a more
aggressive form of CML, such as in accelerated
phase or blast crisis
CML
• It is characterized by a
cytogenetic aberration
consisting of a
reciprocal translocation
between the long arms
of chromosomes 22
and 9 t(9;22)
• The translocation
results in a shortened
chromosome 22
CML
• This translocation relocates an oncogene called
ABL from the long arm of chromosome 9 to a
specific breakpoint cluster region (BCR) in the
long arm of chromosome 22
• The resulting BCR/ABL fusion gene encodes a
chimeric protein with strong tyrosine kinase
activity.
• The presence of BCR/ABL rearrangement is the
hallmark of CML, although this rearrangement
has also been described in other diseases
Lab
• CBC- the increase in mature granulocytes and
normal lymphocyte counts, results in a total
WBC count of 20,000-60,000 cells/Μl
• A mild increase in basophils and eosinophils
is present and becomes more prominent
during the transition to acute leukemia.
• The diagnosis of CML is based on the findings
in the peripheral blood and the Philadelphia
(Ph1) chromosome in bone marrow cells
Lab…
Principles of mx
• The goals of treatment of CML are threefold:
i)Hematologic remission (normal complete
blood cell count [CBC] and physical
examination
ii)Cytogenetic remission (normal chromosome
returns with 0% Ph-positive cells)
iii)Molecular remission (negative polymerase
chain reaction [PCR] result for the mutational
BCR/ABL gene
Principles….
• The chronic phase varies in duration,
depending on the maintenance therapy used:
it usually lasts 2-3 years with hydroxyurea
(Hydrea) or busulfan therapy
• Blast phase is managed as AML
Hypereosinophilic Syndrome
• Its characterized by persistent eosinophilia that
is associated with damage to multiple organs
• Has 3 features:
-A sustained absolute eosinophil count (AEC)
greater than >1500/µl is present, which persists
for longer than 6 months.
-No identifiable etiology for eosinophilia is present.
-Patients must have signs and symptoms of organ
involvement.
BONE MARROW ASPIRATE AND
BIOPSY
Dr. J.C. Kipkulei
Dept of Haematoloty and Blood
Transfusion
Introduction
• Bone marrow examination is useful in the
diagnosis and staging of hematologic disease, as
well as in the assessment of overall bone marrow
cellularity.
• Because of easy accessibility, aspiration, biopsy,
and culture of the bone marrow may also play a
role in the assessment of patients with fever of
undetermined origin as well as in the diagnosis of
various storage and infiltrative disorders.
Background information
• The bone marrow is one of the most widely
distributed organs in the human body.
• It is the principal site of blood formation
beginning at the time of birth, at which time all
bone cavities are filled with hematopoietic tissue.
• By adolescence, active marrow is usually only
found in the cavities of axial bones (sternum, ribs,
vertebrae, clavicles, scapulae, skull, pelvis ) and
the proximal ends of the femurs and humeri
Background information…..
• Overall bone marrow cellularity approximates
100 percent at birth and declines with time,
paralleling an age-associated reduction in
hematopoietic activity.
• Accordingly, bone marrow cellularity in the
normal adult is approximately 50 percent,
with the remainder of the marrow being
composed of adipose tissue
Indications
• Unexplained anemia
• Macrocytic anemia (to distinguish
megaloblastic from normoblastic maturation)
• Unexplained leukopenia
• Unexplained thrombocytopenia
• Pancytopenia
• Presence of blasts on peripheral smear
(investigation for possible leukemia)
Indications….
• Presence of teardrop red cells on peripheral
smear (possible myelofibrosis)
• Presence of hairy cells on peripheral smear
(possible hairy cell leukemia)
• Suspected multiple myeloma
• Staging of non-Hodgkin's lymphoma
• Unexplained splenomegaly (possible
lymphoma)
Indications….
• Suspected storage disease (eg, Gaucher
disease, Niemann-Pick)
• Fever of unknown origin
• Suspected chromosomal disorders in neonates
(requiring rapid confirmation)
• Confirmation of normal marrow in potential
allogeneic donor
• Work-up of amyloidosis (to detect clonal
plasma cell disorder)
Contraindications
Absolute
• Bleeding disorders e.g. hemophilia, severe
disseminated intravascular coagulopathy or
other related severe bleeding disorders
Contraindications….
Relative/precautions
• Patients with suspected multiple myeloma or
other disorders associated with bone resorption
should not undergo sternal bone marrow
aspiration due to an increased risk of sternal
perforation
• Precautions may need to be taken if there is skin
infection or osteomyelitis in the area of proposed
aspiration or biopsy, or if the patient is
uncooperative
Sites of aspiration and biopsy
• Posterior superior iliac crest: This is the most
commonly employed site for reasons of safety,
a decreased risk of pain, and accessibility
• Anterior superior iliac crest: This is an
alternative site when the posterior iliac crest is
unapproachable or not available due to
infection, injury, or morbid obesity
Sites of aspiration only
• The sternum is sampled only as a last resort in
those older than 12 years and in those who
are morbidly obese, but it should be avoided
in highly agitated patients
• The tibia is sampled only for infants younger
than 1 year, and the procedure is conducted
under general anesthesia. This site is localized
to the proximal anteromedial surface, below
the tibial tubercle. The tibial location is not
utilized in older patients because the marrow
Surgical biopsy
• Bone marrow examination may be obtained
by an incisional biopsy at a number of
different bony sites.
• An appropriate site is usually determined by
prior examination of the patient or by the
appropriate radiologic method (e.g, CT scan,
MRI)
• Such procedures may be performed under CT
guidance, as required
Technique and preparations of
samples
• Materials
• Positioning
• Aseptic technique
• Salah and Klima needles for aspiration
• Jamshidi and Islam needle s for biopsy
• Preparattion can be at bedside or the aspirated material
may be anticoagulated and smeared at a later time
• Adequacy of the specimen is determined by the presence
of "spicules," which appear as fatty droplets, granules, or
small chunks of bone, which allow assessment of marrow
cellularity
Complications
• Bleeding
• Infection
• Tumor seeding
• Needle breakage
• Sternal perforation
• Persistent discomfort at site of biopsy
Complications….
• Others- Exceedingly rare complications have
included transient neuropathy with gluteal
compartment syndrome secondary to post-
biopsy bleeding, fracture due to underlying
osteoporosis , and osteomyelitis.
Bleeding and Thrombotic
Disorders
Dr. Lotodo T.L.C
Blood Transfusion
Objectives
1.To describe the process of Hemostasis
2.To classify and describe bleeding disorders
i)Congenital vs acquired
-Vessel wall disorders
-Platelet disorders
-Coagulation factor disorders
3.To describe thrombotic disorders
Hemostasis
• Its a process which causes bleeding to stop,
meaning to keep blood in a fluid state within a
damaged blood vessel
• The response is tightly controlled to prevent
extensive formation of clots and to break
down such clots once damage is repaired
• Hemostatic system represents a balance
between procoagulant and anticoagulant
mechanisms
Hemostasis
• Has five major • Phases
components: -Vascular
-Blood vessels -Platelet
-Platelets -Coagulation
-Coagulation factors -Fibrinolytic
-Coagulation inhibitors
-Fibrinolysis
Hemostasis-Vascular phase
• Immediate
vasoconstriction of
injured vessel
• Reflex constriction of
adjacent arteries and
arterioles
Hemostasis-Platelet Phase
• Adherence of platelets • Released thromboxane
to exposed connective A2, and ADP cause
tissue following a break platelet to aggregate
in endothelial lining • This forms unstable
,potentiated by von primary hemostatic
willebrand factor plug
• Collagen exposure and • Stabilization of the plug
thrombin produced at is achieved when fibrin
the site of injury cause formed by blood
adherent platelets to coagulation is added to
release granules the platelet plug
Hemostasis-Coagulation phase
• Coagulation cascade-
Blood coagulation
which involves
sequential amplification
of coagulation factors
which culminates in
generation of thrombin
• Thrombin converts
soluble plasma
fibrinogen to fibrin
Hemostasis- Fibrinolytic Phase
• Anti-clotting
mechanisms are
activated to allow clot
disintegration and
repair of the damaged
vessel
Bleeding disorders
• Occur due to:
i)Vascular /connective tissue abnormality
ii)Qualitative or quantitative platelet
abnormality
iii)Abnormal coagulation
iv)Defective fibrinolytic system
Classification of bleeding disorders
a)Congenital
i)Vascular/connective tissue
-Hereditary hemorrhagic telengiectasia
-Ehlers-Danlos,Marfans Syndrome
-Osteogenesis Imperfecta
ii)Platelet abnormalities
-Bernard-Soulier
-Wiskott –Aldrich
-May Hegglin anomaly
-Glanzmann thrombosthenia
Classification
iii)Coagulopathies
-Hemophilia A(Factor VIII deficiency) B(factor IX
deficiency),deficiency factor I,V,VII ,IX,XIII
-Von Willebrand disease
-Dysfibrinogenemia
-Plasmin/Plasminogen deficiency or inhibitor
Petechiae, Purpura Hematoma, Joint bl.
Classification…
b) Acquired
i)Vascular/connective tissue
-Vasculitis
-Senile purpura,Henoch-schnolein purpura
-Medications
-Trauma
-Nutritional deficiencies e.g Vit c deficiency
ii)Platelet disorders
-Autoimmune and alloimmune,
-Drug-induced
-hypersplenism
-Thrombotic thrombocytopenic purpura (TTP)
Henoch-Schonlein purpura
20y Male, fever, painful symmetric polyarthritis for a day. During the next two days,
edema and palpable purpura developed.
Classification
• iii)Coagulopathies
-Circulating anticoagulant or inhibitor
-Vit k deficiency-affects factors II,VII,IX,X
iv)Drugs
-Antiplatelet agents-Aspirin
-Anticoagulants-Warfarins
-Antithrombins
-Myelosuppresive drugs
-Hepatotoxic and nephrotoxic drugs
Classification
• V)Hematologic diseases-
-Acute leukemias
-Myelodysplasias,
-Monoclonal gammopathies
VI)Systemic illnesses-renal,liver diseases
VII)Disseminated Intravascular Coagulation- ---
Acute (sepsis, malignancies, trauma, obstetric
complications)
- chronic (malignancies, giant hemangiomas, missed
abortion
EVALUATION-History
• Clinical history
-Presenting complains-site ,duration
,spontaneous/provoked(trauma,sx,dental extraction )
-Past medical history- previous bleeding episodes
,surgery,leukemia
-Family history-hereditary disorders
-Drug history-NSAIDS, Cytotoxic, anticoagulants
-Obs and Gyn history-Abnormal pv bleeding,prolonged
menses
-Nutrition history
EVALUATION-History
Systemic review:
-CNS - Headache, stiff neck, vomiting, lethargy,
irritability, and spinal cord syndromes
-GI - Hematemesis, melena, frank red blood per
rectum, and abdominal pain
-Genitourinary - Hematuria, renal colic, and post
circumcision bleeding
Evaluation-Examination
General examination Epistaxis, oral mucosal
hemorrhage,petechiae,purpura,echymosis,
Weakness ,jaundice,oedema,fever
-Lymphadenopathy,hepatomegaly,splenomegaly
-Musculoskeletal (joints) - Tingling, cracking,
warmth, pain, stiffness, and refusal to use
joint (children)
Laboratory investigations
Components of coagulation screen:
1.Complete blood count(Hb, WBC,Platelets),
2.Peripheral blood film
3.Bleeding time
4.Clotting time
5.Activated partial thromboplastin
time(aPTT)/Kaolin Cephalin clotting time(KCCT)
6 .Prothrombin time
7 .Thrombin time
Laboratory…
• Further tests:
i)Investigation of fibrinolytic mechanism-Factor
degradation assay
ii)Biochemistry-LFT’S, Renal functions
iii)Substitution tests
iv)Specific factor assays
v)Platelet function tests
Laboratory
• Specimens required:
i)5ml of blood in EDTA for CBC,PBF
ii)4.5ml of blood in 0.5ml of 3.2 citrate for
coagulation studies
iii)5ml of blood in Trasylol for FDP assay
Bleeding time(ivy’ method)
• Principle: Duration of bleeding from standard
cut is measured. Bleeding depends on
elasticity of blood vessel wall and upon the no
and function integrity of platelets
• Average bleeding time is 2-7mins
Prothrombin Time (PT)
Principle- assesses extrinsic pathway
- It measures the presence and activity of five different
blood clotting factors (factors I, II, V, VII, and X). –
- Tissue thromboplastin and calcium is added to
citrated plasma
Results.
– When using the Prothrombin time for oral
anticoagulant control(warfarin), the result should
also be expressed as the International Normalized
Ratio (INR).
– -Normal time is 13-16s
Interpretation
• Normal Range - within 2 seconds of control time.
• A prolonged prothrombin time may be caused by:
– Deficiency of factors I,11, V, VII,X or inhibitors to this
coagulation factors
– Inherited defects or acquired due to consumption (e.g.
DIC),
– Liver disease (vitamin K deficiency or lack of synthesis)
– Factor dilution (massive transfusion)
– Anticoagulants use (warfarin)
• If no clot is seen after 180 seconds the test should be stopped
and reported as > 180 seconds.
ACTIVATED PARTIAL THROMBOPLASTIN TIME
(APTT)/KCCT
Principle
-Assesses intrinsic pathway(VIII,IX.XI,XII)
– This test derives its name from the use of a partial
thromboplastin, or "cephalin" which is a
phospholipid component that is added to a
specimen that has been "activated" by exposure to
a negatively charged substance (kaolin)
– Normal range 30 – 40 seconds
Interpretation:
– A prolonged APTT may be caused by:

a) Deficiency of factors II, V, VIII, IX,X, XI,
XII,fibrinogen due to :
– Inherited defects (e.g. hemophilia)
– Acquired because of consumption e.g.
» DIC,
» Liver disease (Vitamin K deficiency or
decreased synthesis),
» Dilution (massive transfusion), or
» Anticoagulant (coumarin derivatives)
b) Heparin therapy
c) Specific factor inhibitors
Thrombin Time
Principle
– The TT measures the rate of fibrin clot formation
after the addition of a standard concentration of
thrombin to citrated plasma
– It assesses deficiency or abnormality of
fibrinogen or inhibitor of thrombin
– Normal 14-16s
Interpretation.
• Normal range - within 2 seconds of control time.
• A Prolonged thrombin time may be caused by:
1. Deficiency of fibrinogen - inherited(
afibrinogenaemia or hypofibrinogenaemia) or
acquired.
2. Abnormal fibrinogen molecule
(dysfibrinogenaemia)- inherited or acquired.
3. Raised levels of FDPs.
4. Presence of heparin or heparin -like substances in
the sample
Management
• Supportive
-ABC-Colloids ,Chrystalloids
• Pain – paracetamol, tramal, codeine, avoid
NSAIDS
• Antifibrinolytics e.g.tranexamic acid, ε-
aminocaproic acid. Avoid in genitourinary
bleeding- can cause obstruction and anuria
• DDAVP (desmopressin)
Management
Specific
• Fresh frozen plasma- 1 unit of FFP increases most
coagulation factors by 3% in an adult without DIC
• Platelet concentrates- 1–2 U/10 kg body weight are
sufficient for most DIC patients with severe
thrombocytopenia.
• Cryoprecipitate - rich in FVIII, fibrinogen, vWF
,fibronectin also FXIII
• FVIII concentrates
– Plasma derived
– Recombinant
Management
• Genetic
– Genetic counseling
– Prenatal diagnosis – Carrier detection
– Factor assays
– DNA probes- chorionic biopsies
Hemophilia A
• Hemophilia A is an inherited, X-linked, recessive
disorder
• Its caused by deficiency of functional plasma
clotting factor VIII (FVIII), dysfunctional factor VIII,
or factor VIII inhibitors
• This leads to the disruption of the normal
intrinsic coagulation cascade
• Resulting in spontaneous hemorrhage and/or
excessive hemorrhage in response to trauma.
• Sometimes may occur spontaneously (acquired)
Hemophilia A
• The hallmark of hemophilia is hemorrhage into
the joints.
- Bleeds affect weight-bearing joints and other
joints, resulting in permanent deformities,loss of
mobility, and extremities of unequal lengths
- Muscles most commonly affected are the flexor
groups of the arms and gastrocnemius of the
legs.
- Ilio-psoas bleeding is dangerous because of the
large volumes of blood loss and because of
compression of the femoral nerve
HEMOPHILIA A
HEMOPHILIA A
• Persons with less than 1% normal factor (<
0.01 IU/mL) have severe hemophilia.
- 1-5% normal factor (0.01-0.05 IU/mL) are
considered to have moderately severe
hemophilia.
- Persons with more than 5% but less than 40%
normal factor (>0.05 to < 0.40 IU/mL) are
considered to have mild hemophilia
HEMOPHILIA A
• Management is through multi-displinary
approach
• Appropriate education and treatment is required
in patients with hemophilia
• Prophylaxis and early treatment with FVIII
concentrate that is safe from viral contamination
have dramatically improved the prognosis of
patients
• Regular dental evaluation is recommended, along
with instruction regarding proper oral hygiene,
dental care, and adequate fluoridation.
HEMOPHILIA A
• Advise the patient against participating in
contact and collision sports.
• Patient and family education about early
recognition of hemorrhage signs and symptoms is
important for instituting or increasing the
intensity of replacement therapy.
• This treatment helps prevent the acute and
chronic complications of the disease that may
vary from life-threatening events to quality-of-
life–impairing events.
Platelet disorders
• Platelets arise from the fragmentation of the
cytoplasm megakaryocytes in the bone marrow
• Circulate in blood as disc-shaped anucleate
particles.
• Platelet disorders lead to defects in primary
hemostasis
• Primary hemostatic disorders are characterized
by prolonged bleeding time, and the
characteristic physical examination findings are
petechiae and purpura.
Thrombocytopenia
• Results from :
i)Decreased bone marrow production
-Bone marrow failure such as aplastic anemia
-Infiltration by leukemia or another malignancy
- Fibrosis, granulomatous disorders, or tuberculosis.
ii)Increased platelet destruction
-Immune mechanism e.g ITP
-Increased consumption e.g DIC
-Drug induced
THROMBOCYTOPENIA
iii)Disorders of platelet function
-Disorders of platelet adhesion (von Willebrand
disease, Bernard-Soulier syndrome)
-Disorders of aggregation (Glanzmann
thrombasthenia)
-Morphological anomalies-Chediak Higashi,May
Hegglin anomaly
-Acquired disorders of platelet function (drugs [eg,
aspirin, NSAIDs, alcohol])
-Uremia
Disseminated Inravascular coagulation
(DIC)
• Disseminated intravascular coagulation (DIC)
is a complex systemic thrombo-hemorrhagic
disorder
• Involves the generation of intravascular fibrin
and the consumption of procoagulants and
platelets.
• The resultant clinical condition is
characterized by intravascular coagulation and
hemorrhage
DIC
• Components of DIC include the following:
• Exposure of blood to procoagulant substances
• Fibrin deposition in the microvasculature
• Impaired fibrinolysis
• Depletion of coagulation factors and platelets
(consumptive coagulopathy)
• Organ damage and end organ failure
DIC
• Conditions associated with disseminated intravascular
coagulation include the following
• Sepsis/severe infection,trauma (neurotrauma)
• Malignancy (solid and myeloproliferative malignancies)
• Rheumatologic illness - Adult Stills disease, lupus
• Obstetric complications -Amniotic fluid embolism abruptio
placentae hemolysis, elevated liver enzymes, low platelets
(HELLP) syndrome/eclampsia
• Retained dead fetus syndrome
• Vascular abnormalities -Kasabach-Merritt syndrome, large
vascular aneurysms
• Severe toxic reactions - Envenomations, transfusion
reactions, transplant rejection
• Heat stroke/hyperthermia
Thrombotic disorders
• Abnormal thrombosis-could be arterial or venous
• Deep venous thrombosis (DVT) and pulmonary
embolism (PE) are manifestations of a single disease
entity, namely, venous thrombo-embolism (VTE).
• DVT is the presence of coagulated blood, a thrombus,
in one of the deep venous conduits that return blood
to the heart
• However, if left untreated, the thrombus may become
fragmented or dislodged and migrate to obstruct the
arterial supply to the lung, causing potentially life-
threatening PE
Thrombosis Risk Factors
Endothelial damage
• Atherosclerosis
Stasis
• Immobilization
• Varicose veins
• Cardiac dysfunction
Hypercoagulability
• Surgery
• Carcinoma
• Estrogen/postpartum
• Thrombotic disoders
Deep venous thrombosis
Pulmonary embolus
DVT
• Objectives for the treatment of deep venous
thrombosis (DVT) are to:
- Prevent pulmonary embolism (PE)
- Reduce morbidity
- Prevent or minimize the risk of developing
the post-thrombotic syndrome (PTS
• Mainstay of medical therapy has been
anticoagulation since the introduction of
heparin in the 1930s
DVT
• Other anticoagulation drugs such as vitamin K
antagonists and low-molecular-weight heparin
(LMWH) have been used subsequently.
• More recently, mechanical thrombolysis has
become increasingly used as endovascular
therapies have increased.
• Absolute contraindications to anticoagulation
treatment include :intracranial bleeding, severe
active bleeding, recent brain, eye, or spinal cord
surgery, pregnancy, and malignant hypertension.
Summary
• Bleeding and thrombotic disorders may
present as medical emergencies
• Evaluation of patient involve taking complete
hx, thorough physical examination and
directed laboratory findings to find the
underlying cause
• Proper management includes: supportive
care,specific treatment,proper follow up and
rehabilitation
LABORATORY EVALUATION OF
PATIENTS WITH BLEEDING
DISORDERS
Dr. J.C. Kipkulei
Dept of Haematology and Blood
Transfusion
OVERVIEW OF HEMOSTASIS
Dependent upon:
– Vessel Wall Integrity
– Adequate Numbers of Platelets
– Proper Functioning Platelets
– Adequate Levels of Clotting Factors
– Proper Function of Fibrinolytic Pathway
Intrinsic Extrinsic
Collagen Tissue Thromboplastin
XII
XI V vII
IX
VIII
V FIBRINOGEN
(I) (I)
PROTHROMBIN THROMBIN
(II) (III) FIBRIN
LABORATORY TESTS
1) CBC/Platelet count
2) Bleeding time
3) Prothrombin time
4) INR
5) Partial thromblastin time
6) Fibrin D-dimer
7) Clot solubility
8) Assays of clotting factor VIII, IX , von Willebrand
factor
Obtaining the Blood Sample
• A properly drawn blood sample is the key to
interpreting the results of clotting tests
• Whole blood is collected into anticoagulant,
using an evacuated sample tube containing a
fixed amount of anticoagulant, in the ratio of
one part of anticoagulant solution to nine
parts of whole blood
• The anticoagulant of choice for coagulation
studies is trisodium citrate
Obtaining the Blood Sample….
• The sample must be free of tissue fluids,
intravenous solutions delivered through
indwelling lines, and heparin
• The anticoagulated blood should be mixed
gently by inversion three or four times, sent to
the laboratory in an expeditious manner, and
tested within two hours if kept at room
temperature (22 to 24ºC) or within four hours
if kept cold (2 to 4ºC).
1. PLATELET COUNT
• NORMAL 150,000 - 400,000 CELLS/MM 3
• < 150,000 Thrombocytopenia
• 50,000 - 150,000 Mild Thrombocytopenia

• 20,000 – 50,0000 Moderate Thrombocytopenia
• <20,000 severe thrombocytopenia

2. BLEEDING TIME
• Used to test for platelet function

• Normal range: 2-9 minutes depending on the
method
– Ivy method- less than 9.5 minutes
– Duke method- 1-3 minutes
Causes of prolonged bleeding time
• Thrombocytopenia
• Disorders of platelet function- Bernard-Soulier disease,
Glanzmann's thrombasthenia and drugs(aspirin and
other cyclooxygenase inhibitors)
• Disseminate intravascular coagulopathy( DIC)
• von Willebrand disease
• Vascular abnormalities e.g. Ehrer-Danlos’s syndrome
• Hypofibrinogenemia
• Occasionally in severe deficiency of factor V and XI
3. PROTHROMBIN TIME(PT)- normal value 10-15
seconds
• Uses:
To assess the extrinsic pathway of clotting, which
consists of tissue factor and factor VII, and
coagulation factors in the common pathway
(factors II [prothrombin], V, X, and fibrinogen)
 To monitor warfarin therapy
• Mnemonic- PET
Causes of prolonged PT
• Warfarin therapy
• Vitamin K deficiency due to e.g. poor nutrition
or use of broad spectrum antibiotics
• Liver disease
• Deficiency or inhibition of factors VII, X, II, V or
Fibrinogen
• The infrequent antiphopholipid
antibodies(Lupus anticoagulant phenomenon)
3. INTERNATIONAL NORMALIZED RATIO(INR)
• The INR is calculated from the following

formula:
INR = [Patient PT ÷ Control PT]
• It is used to monitor warfarin therapy
• The therapeutic range for the INR varies with
the clinical indication. For most indications the
recommended range is 2.0 to 3.0
4. ACTIVATED PARTIAL THROMBLASTIN TIME
(aPTT)- normal range 25-40 seconds
• Uses:
To assess the integrity of the intrinsic coagulation
pathway (prekallikrein, high molecular weight
kininogen, factors XII, XI, IX, VIII) and final
common pathway (factors II, V, X, and fibrinogen)
To monitor heparin therapy
• Mnemonic- PITT
Causes of prolonged aPTT
• Prolongation of the aPTT can occur with a

deficiency of, or an inhibitor to, any of the
clotting factors except for factor VII
aPTT in monitoring Heparin therapy
• Heparin is an indirect thrombin inhibitor which

complexes with antithrombin (AT), converting this
circulating cofactor from a slow to a rapid inactivator of
thrombin, factor Xa, and to a lesser extent, factors XIIa,
XIa, and IXa
• The critical therapeutic level of heparin to be reached
within 24 hours (as measured by the aPTT) is 1.5 times
the mean of the control value or the upper limit of the
normal aPTT range.
• The goal of maintenance heparin therapy is to maintain
the aPTT in the range of 1.5 to 2.5 times the patient's
aPTT baseline value
5. THROMBIN TIME- normal 7-13 seconds
• The thrombin time (TT) measures the final

step of the clotting pathway, the conversion of
fibrinogen to fibrin.
Causes of prolonged TT
• Presence of heparin
• Presence of fibrin or fibrinogen degradation
products
• Hypofibrogenemia(< 100mg/dl),
dysfibrinogenemia or hyperfibrogenemia (>
400 mg/dl)
• High concentration of serum proteins as in
cases of multiple myeloma and amyloidosis
6. FIBRIN D-dimer
• Plasmin cleaves fibrin at multiple sites and

releases fibrin degradation products (FDPs). One
of the major FDPs is D-dimer, which consists of
two D domains from adjacent fibrin monomers
that have been crosslinked by activated factor
XIII.
• Since D-dimer is generated from cross-linked
fibrin, but not from fibrinogen, an elevated
plasma concentration of D-dimer indicates recent
or ongoing intravascular blood coagulation
Causes of increased D-dimer
• Arterial thromboembolic disease

• Venous thromboembolic disease
• Disseminated intravascular coagulation
• Preeclampsia and eclampsia
• Abnormal fibrinolysis; use of thrombolytic agents
• Cardiovascular disease, congestive failure
• Severe infection/sepsis/inflammation
• Surgery/trauma (eg, tissue ischemia, necrosis)
• Systemic inflammatory response syndrome
• Vasoocclusive episode of sickle cell disease
7. CLOT SOLUBILITY TEST
• Used to assess deficiency of factor XIII

• Clots formed in the presence of factor XIII and
Ca 2+ are stable (due to cross linking) whereas
clots formed in the absence of factor XIII
dissolve.
ERYTHTOCYTE SEDIMENTATION
RATE (ESR)
Dr. J.C.Kipkulei
transfusion
Definition
• ESR is the rate at which RBCs sediment in a period of
1hr
• Is a common screening hematologic test that is a non-
specific measure of inflammation
Basis
• ESR is governed by the balance between pro-
sedimentation factors- fibrinogen and other acute
phase proteins( haptoglobin, ceruloplasmin, α1-
antitrypsin, α1-acid glycoprotein, c-reactive protein)
and anti-sedimentation factors- negative charge of
RBCs and albumin
Basis….
• When an inflammatory process is present, the
high concentration of acute phase proteins in
the blood causes the RBCs to stick together
• The RBCs form stacks called ‘rouleaux’ which
settle faster
Method
• Anticoagulated blood is placed in an upright tube,
known as a Westergren tube, and the rate at
which the red blood cells fall is measured and
reported in mm/h.
Dynamics
• ESR rises 24hrs after inflammation onset and
symptoms
• Gradually returns to normal 4 weeks after
resolutioon
Normal Reference range
• Newborn: 0-2mm/hr
• Children: 0-13mm/hr
• Women
– 18-50yrs: 0-20mm/hr
– >50yrs: 0-30mm/hr
– Normal maximum: (Age in yrs + 10)/2
• Men
– 18-50yrs: 0-25mm/yr
– 50yrs: 0-20mm/yr
– Normal maximum: Age in yrs/2
Factors affecting ESR
• Older age
• Female gender
• Pregnancy
• Anemia
Indications
ESR is done to:
• Find out if inflammation is present- multiple myeloma,
temporal arteritis, polymalgia rheumatica, SLE,
rheumatoid arthritis, TB, rheumatic fever etc
• Check on the stage/grade/progress of disease-
inflammatory bowel disease in children, rheumatic
arthritis, osteomyelitis, pneumonia, pyelonephritis etc
• Monitor on the response to treatment in certain
inflammatory diseases- temporal arteritis, polymyalgia
rheumatica, rheumatoid arthritis, Hodgkin’s lymphoma
etc
CAUSES OF INCREASE IN ESR
• Infectious Disease
– Bacterial Infections
– Infectious hepatitis
– Cat Scratch Disease
– Post-perfusion syndrome
– Primary Atypical Pneumonia
– Tuberculosis
– Secondary Syphilis
– Leptospirosis
– Systemic Fungal Infection
• Hematologic and Neoplastic Disease
– Severe Anemia or macrocytosis
– Leukemia
– Lymphoma
– Metastatic tumor
– Chronic granulomatous disease
• Gastrointestinal Disease
– Inflammatory Bowel Disease
– Acute Pancreatitis
– Lupoid hepatitis
– Cholecystitis
– Peritonitis
• Collagen Vascular Disease
– Rheumatic Fever
– Rheumatoid Arthritis
– Systemic Lupus Erythematosus
– Dermatomyositis
– Scleroderma
– Systemic Vasculitis
– Henoch-Schonlein Purpura
– Mediterranean fever
• Renal disease
– Acute Glomerulonephritis
– Chronic Glomerulonephritis with Renal Failure
– Nephrosis
– Pyelonephritis
– Hemolytic Uremic Syndrome(HUS)
• Miscellaneous disorders
– Hypothyroidism
– Thyroiditis
– Sarcoidosis
– Infantile cortical hyperostosis
– Trauma from surgery or burn injury
– Drug Hypersensitivity Reaction
PERIPHERAL BLOOD FILM
Dr. J.C. Kipkulei

Transfusion
• A.k.a blood film or peripheral blood smear
• Is a thin layer of blood spread on a microscope
slide and stained in such a way to allow the
various cells to be examined microspically
• PBF is used to supplement information
obtained from CBC
• PBF is required for precise classification of
abnormal blood cells
INDICATIONS
• Thin film: To investigate/evaluate
haematological disorders e.g. anemia,
leukemia
• Thick film: To look for parasites e.g. malaria,
filaria, babesia, Leishmania spp, Trypanosoma
spp, Ehrlichilosis
PREPARATION OF THIN FILM
• The technique used is wedge slide(‘push
slide’) developed by Maxwell Wintrobe
• A drop of blood is placed on one of slide
• A spreader slide is then used to disperse the
blood over the slide length
• Slide is left to air dry then fixed in methanol
• Slide is finally stained with a dye e.g. Wright,
Giemsa, Leishman etc and examined
Step 1. Placing a small
drop of venous blood
on a glass microscope
slide, using a glass
capillary pipette. A
wooden applicator stick
can also be used for this
purpose.
Step 2. A spreader slide
has been positioned at
an angle and slowly
drawn toward the drop
of blood
Step 3. The spreader slide
has been brought in
contact with the drop of
blood and is being
drawn away. Note layer
of blood at the edge of
the spreader slide.
Step 5. The blood smear is
nearly complete.
Step 6. End result. A glass
slide with a well-formed
blood film. After drying
for about 10 minutes,
the slide can be stained
manually or placed on
an automated slide
stainer.
Fig. 2. Photograph of the
peripheral blood smear
prepared above. The
arrow points to the
zone of morphology.
EVALUATION OF PBF- Morphology of
RED BLOOD CELLS
• Normal RBC- ("normocytes," "discocytes") are
round to very slightly ovoid cells with a mean
diameter of approximately 7 mm and a central
pale area ("area of central pallor") approximately
1/3 the diameter of the cell that gradually fades
towards the more deeply stained periphery.
• The RBC is approximately the same size as the
nucleus of a mature lymphocyte.
• Any deviation in size, volume, shape or presence
of inclusions represents an abnormal red blood
cell.
Clinical Importance of RBC Morphology
• Since different types of abnormal red blood
cells arise by different etiologic processes,
disease diagnosis can often be made by
interpretation of red blood cell pathology in
conjunction with CBC data and other clinical
and laboratory information
Classification of RBC Morphologic Abnormalities
Hypochromia
• Less dense staining of RBC due to decreased
amount(MCH) and concentration(MCHC) of
hemoglobin
• In the peripheral blood smear, hypochromic cells
have an expanded central zone of pallor
• Seen in iron deficiency anemia, thalassaemia,
sideroblastic anemia, lead poisoning
Hyperchromia
• More dense staining of RBC due to high
concentration of RBC Hb(MCHC)
• Seen in hereditary spherocytosis
Microcytes
• Are small red blood cells (MCV < 8O fL) with
decreased amounts of hemoglobin formed
• Seen in iron deficiency anemia, thalassaemia,
lead poisonig, anemia of chronic disease,
sideroblastic anemia
Round macrocytes
• Are slightly to moderately larger than normal
red cells(MCV >100 fL but usually < 120 fL)
and are round in shape
• Seen in liver disease, drugs(chemotherapy),
myeloproliferative disorders, myelodysplastic
synrome, splenectomy
Oval macrocytes ("macroovalocytes,
megalocytes")
• Are large oval red blood cells (> 8.5 mm) with
an elevated MCV (> 100 fL, frequently > 120
fL) and normal MCH
• Seen in vitamin B12 and folate deficiency
Anisocytosis
• Variation in RBC size
• Seen in meglablastic anaemia, thalassaemia
and iron deficient anaemia
Poikilocytosis
• Variation in RBC shape
• Seen in iron deficient anaemia, myelofibrosis,
thalassaemia
Polychromasia
• Is the occurrence of slightly immature red
blood cells, which are larger than normal
(increased MCV) and have a blue-gray
coloration
• This is a response to bleeding, haemolysis or
marrow infiltration
Sickle cells (‘Drepanocytes’ )
• Are curved, irregular red blood cells with pointed ends
• Seen in sickle cell enemia and other ‘sickle
hemoglobinopathies
Acanthocytes
• Acanthocytes ("spur cells, spicule cells") are spheroid
RBCs with a few large spiny (thorny) projections due to
an unstable red cell membrane lipid structure
• Seen in abetalipoproteinemia; can be seen also after
splenectomy, alcoholic cirrhosis, hemolytic anemias
Bite cells
• Bite cells (degmacytes) are RBCs with
peripheral single or multiple arcuate defects
("bites").
• Seen in G6PD and pyruvate kinase deficiency
Target cells
• Target cells ( ‘codocytes’) are thin,
hypochromatic cells with a round area of
central pigmentation
Burr cells ("crenated cells")
• Are red blood cells with multiple tiny spicules (10-
30) evenly distributed over the cell surface
• Seen in uremia, where they represent damaged
or fragmented red blood cells.
Tear drop cells (‘dacrocytes’)
• Are red blood cells in the shape of a teardrop
• Seen in myelofibrosis, megaloblastic anemia, B-
thalassaemia, acuired hemolytic anemia, renal
disease hyperplenism
Elliptocytes
• Elliptocytes are cells with an elliptical shape
• Seen in hereditary elliptocytosis
Keratocytes/ schistocytes
• Keratocytes ("horn cells, helmet cells") and
schistocytes ("fragmented cells") are damaged red
blood cells
• Seen in DIC, microangiopathic hemolytic anemia,
thrombotic thrombocytopenic purpura (TTP),
prosthetic heart valves, severe valvular stenosis,
malignant hypertension, or march hemoglobinuria
Spherocytes
• Spherocytes are small (< 6.5 mm), dense
spheroidal RBCs with normal or decreased
MCV and absent central pallor
• Seen in hereditary spherocytosis, autoimmune
hemolytic anemia, microangiopathic
hemolytic anemia, hypersplenism and post-
splenectomy, myelofibrosis with myeloid
metaplasia, hemoglobinopathies, malaria,
liver disease, recent transfusions, and severe
Stomatocytes
• Stomatocytes are uniconcave red blood cells
with a slit-like area of central pallor.
• Seen in hereditary stomatocytosis (a type of
hemolytic anemia).
Reticulocytes
• Young/immature, larger RBCs signifying active
erythopoiesis
• Increased in haemolysis and haemorrhage
Basophilic stippling
• Basophilic stippling is the occurrence of fine,
medium, or coarse blue granules uniformly
distributed throughout some red blood cells
• Seen in heavy metal poisoning (e.g. lead and
arsenic), hemoglobinopathies, thalassemias,
sideroblastic anemias
Howell-Jolly bodies
• Are small (1 mm) dense, perfectly round
basophilic red cell inclusions which represent
nuclear material derived from nuclear
fragmentation ("karyorrhexis) or incomplete
nuclear expulsion during normoblastic
maturation
• Seen after splenectomy
Pappenheimer bodies
• Granules of siderocytes containing iron
• Seen in lead poisoning, carcinomatosis, post-
splenectomy
Heinz bodies (Heinz-Hehrlich bodies)
• Are inclusions within RBC composed of denatured
hemoglobin
• Seen in G6PD, α-thalassaemia, chronic liver
disease
Rouleaux formation
• Rouleaux formation ("pseudoagglutination") is
a linear arrangement of RBCs ("coinstack")
caused by an increased blood concentration of
fibrinogen, globulin, or paraproteins
• Seen in acute and chronic inflammatory
disorders, Waldenstrom’s macroglobulinemia,
and multiple myeloma
EVALUATION OF PBF- WBC
Classification of WBC Morphologic
Abnormalities
Alder-Reilly granules
• Alder-Reilly granules are large, coarse, dark
purple, azurophilic granules that occur in the
cytoplasm of most granulocytes
• Seen in Alder-Reilly anomaly and in patients
with mucopolysaccharidoses.
Auer rods
• Auer rods are needle- or rod-shaped, eosinophilic
structures which occur in the cytoplasm of immature
leukocytes (blasts) and more mature cells in some
patients with acute myelogenous leukemia (AML)
• Seen in AML FAB subtypes M1, M2, M3 and M4
Chédiak-Higashi granules
• Chédiak-Higashi granules are very large red or blue
granules that appear in the cytoplasm of granulocytes,
lymphocytes, or monocytes in patients with the
Chédiak-Steinbrinck-Higashi syndrome.
Döhle bodies
• Döhle bodies are variably sized (0.1 to 2.0 um)
and shaped, blue or grayish-blue cytoplasmic
inclusions usually found near the periphery of
the cell.
• Dohle bodies are lamellar aggregates of rough
endoplasmic reticulum, which appear in the
neutrophils, bands, and metamyelocytes of
patients with infection, burns, uncomplicated
pregnancy, toxic states, or during treatment
Neutrophilic hypergranulation (toxic
granulation)
• Small dark blue to purple granules resembling
primary granules appear in the cytoplasm of
metamyelocytes, bands, and segmented
neutrophils
• Seen in sepsis and other inflammatory states,
burns, and trauma, and upon exposure to
hematopoietic growth factors such as
granulocyte-colony stimulating factor (G-CSF).
Neutrophilic hypersegmentation
• Increased lobulation of granulocyte nuclei
(neutrophilic hypersegmentation)
• Seen in megaloblastic anemia or as an inherited
autosomal dominant trait (hereditary
hypersegmentation of neutrophils).
Blasts
• Nucleated precursor cells
• Seen in myelofibrosis, leukaemia, malignant
infiltration by carcinoma
Leukemoid reaction
• Marked leucocytosis with significant increase
in neutrophil precursors( myelocytes,
metamyelocytes and promyelocytes)
• Is a physiologic response to severe illness
(stress or infection)
Leucoerythroblastic anemia
• Anemia characterized by appearance of
immature myeloid and nucleated erythrocytes
• Seen in infiltration of bone marrow by
malignancy, anorexia, sepsis and sever
hemolysis
Left shift/blood shift
• An increase in the number of immature WBC
in the peripheral blood, particularly neutrophil
band cells
ORAL MANIFESTATIONS OF
HEMATOLOGIC DISORDERS.
• ANAEMIA: Refers to state in which patients
blood hemoglobin is below the normal range
for patients age and sex.
male 13.6-17.2 g/dl

Women 12.0-15.0 g/dl
Manifests orally as Pallor of the oral mucosae

angular cheilitis,smooth tongue.
JAUNDICE
• Yellow appearance of the skin, mucous

membranes and sclerae resulting from
increased bilirubin concentration in body
fluids.
• Normal range 3-17 micro mol/ l
Detectable at a concentration of 50 micro mol/l
[3g/dl].
Bilirubin do not cross BBB in adults but can cross
in immediate neonatal life.
BLEEDING DISORDERS
hereditary bleeding disorders.
• Hemophilia: represents a variety of bleeding disorders
associated with a genetic deficiency of one of the
clotting factors of blood.
Hemophilia A:[factor 8 deficiency]:Accounts for 80-85%
of bleeding diathesis.
Hemophilia B:[factor 9 deficiency].
X- linked recessive hereditary disease
• Von willebrand disease: genetic deficiency of plasma
glycoprotein. Most common of inherited bleeding
disorders [1 in 800 or 1000]
• Severity depends on the extent of clotting factor
deficiency.
Oral manifestations:
• Oral ulcerations, lip and tongue ecchymoses.
• Hemarthrosis in joints TMJ,knee joints which
leads to crippling deformity secondary to arthritis
and ankylosis.
2.thrombocytopenia: reduced circulating platelets.
Necessary for hemostasis and clotting.Normal
range 200,000-400,000/mm3 of blood.
• Causes :reduced production,increased
destruction or sequestration in spleen.
Oral manifestation:
• Manifests when platelets fall below
100,000/mm3 of blood.
• Pin point hemorrhagic
lesions[petechiae],ecchymosis and haematoma.
Management :
If drug-related stop administration.
Platelet transfusion.
NEUTROPENIA
• Reduced circulating neutrophils [below

1500/mm3 of blood.
• Manifests with increased susceptibility to
bacterial infections if other elements of immune
system e.g lympocytes,macrophages and plasma
cells are intact ; gingival ulcerations and these
ulcers characteristically lack erythematous
periphery.
• Histopathology: reduced or absence of
neutrophils.
LEUKAEMIA.
• Represents malignancies of hematopoietic stem

cell derivatives.
• Classified as acute or chronic myeloid or
lymphocytic/lymphoblastic leukaemias.
• Manifests as oral ulcerations,occasionally
leukaemic cells infiltrate oral soft tissue n
produce boggy non tender swelling that may or
may not ulcerate.-tumor-like collection of
leukaemic cells is called granulocytic
sarcoma/extramedullary myeloid tumor.
BURKITTS LYMPHOMA.
• Malignancy of B-lymphocyte that represents
undifferentiated lymphoma.
• Named after Dennis Burkitt[missionary doctor].
• Classified to African and American[sporadic]
burkitts lymphoma.
• Seem to have predilection in jaws.[50-70%] and
children peak age 7 yrs.
• Associated to EBV.90% of tumors have EBV
nuclear antigen and affected pts have elevated
A/B titre to EBV.
Manifestations.
Facial
swelling,proptosis,pain,paraesthesia,marked
teeth mobility.
Hodgkins lymphoma.
• Most authorities classify it as
lymphoproliferative disorder,although poorly
understood.
• Neoplastic cells [reed-sternberg cells] makes
up 1-3 % of cells in enlarged lymph nodes.
• Recent evidence points these cells to B-
lymphocyte origin.
• Oral manifestations rare.
Non –hodgkins lymphomas.
• Includes diverse n complex gp of malignancies
of lymphoreticular histogenesis.
• Mostly arise in lymph node,grow as solid mass
as opposed to lymphocytic leukaemias which
begin in BM N characterized by large
proportion of malignant cells that circulate in
peripheral blood.
• Originate in cells of B- lymphocyte origin.
Tumors of T- cells are less common.
manifestations
• Orally as extra nodal disease.
• May manifest in oral soft tissues or centrally in
the jaw.soft tissue lesion appear as non-tender
diffuse swellings.mostly affect buccal
vestibule,posterior hard palate or gingiva-have
boggy consistency.
• Lesions may appear erythematous or purplish
and it may or may no ulcerate.
• Lymphoma of the bone may cause vague pain
which may b mistaken 4 toothache.paraeshesia.
COMPLETE BLOOD COUNT
Dr. J.C. Kipkulei

Transfusion
• Synonyms:
– Full blood count
– Full hemogram
– Full blood exam
– Blood panel
• It is a blood test done to assess the patients’
blood cells:
– RBCs(erythrocytes)
– WBCs(leukocytes)
– Platelets(thrombocytes)
BASIS
• CBC is among the most commonly performed
blood tests in medicine
• Often performed as a baseline/screening test
in the initial assessment of patients
• It provides an overview of a patients general
health status
• Abnormally high or low counts may indicate
the presence of many forms of disease
METHODS
• Blood is collected in test tubes containing
anticoagulants e.g. Ethylenediamine
tetraacetic acid(EDTA) or sometimes citrate
• The count is either done manually(by a blood
film examined under microscope- Nuebauer
chamber) or by an automated analyzer(e.g.
Beckman Coulter counter)
COMPONENTS OF CBC
• A complete blood count will normally include:
1. RED CELLS
a) Total red blood cells— The number of red cells is
given as an absolute number per litre.
b) Hemoglobin - The amount of hemoglobin in the
blood, expressed in grams per decilitre. (Low
hemoglobin is called anemia.)
c) Hematocrit or packed cell volume (PCV) - This is
the fraction of whole blood volume that consists
of red blood cells.
Hct (L/L, %) = RBC (cells/L) x MCV (L/cell)
COMPONETNS……..
d) Red blood cell indices
– Mean corpuscular volume (MCV) - the average
volume of the red cells, measured in femtolitres.
MCV = Hct
Total RBC
Anemia is classified as microcytic or macrocytic
based on whether this value is above or below the
expected normal range.
– Mean corpuscular hemoglobin(MCH) - the average
amount of hemoglobin per red blood cell, in
picograms.
– Mean corpuscular hemoglobin

concentration(MCHC) - the average concentration
of hemoglobin in the cells.
• Red blood cell distribution width (RDW) - a
measure of the variation of the RBC
population
COMPONENTS…..
2. WHITE CELLS
a) Total white blood cells - All the white cell types
are given as a percentage and as an absolute
number per litre.
b) White blood cell differentials- absolute or
relative
– Neutrophils( also known as segs, PMN, granulocytes)
– Lymphocytes,
– Monocytes
– Eosinophils
– Basophils
COMPONENTS…..
3. PLATELETS
a) Platelet numbers are given, as well as
information about their size and the range of
sizes in the blood.
b) Mean platelet volume(MPV) - a
measurement of the average size of platelets.
NORMAL REFERENCE RANGE
TEST PATIENT NORMAL UNIT COMMENT

RANGE
RED BLOOD Male 4.3-6.2 X 106/µL or
CELLS(RBC) X1012/L
Female 3.8-5.5 ,,
Infant/child 3.8-5.5 ,,
Hemoglobin Male 13-16 mg/dl Higher in
neonates than
in children
Female 12-15 mg/dl Sex difference
negligible until
adulthood
TEST PATIENT NORMAL UNIT COMMENT
RANGE
Hematocrit Male 39-54 %
Female 36-48 %
Child 31-43 %
MCV Male/Female 76-100 Femtolitre(fl) Cells are larger
in neonates,
though smaller
in other
children
MCH Male/Female 25-35 Picogram(pg)
MCHC Male/Female 31-35 Mg/dl
RETICULOCYTE Adult 0.5-1.5 %
Infant 0.5-3.1 %
Newborn 1.1-4.5 %
TEST PATIENT NORMAL RANGE UNIT
WHITE BLOOD CELL Adult 4-11 x109 /dl or x103
COUNT(WBC) /mm3 or x103/µL
Newborn 9-30 ,,
1 year 6-18 ,,
WBC DIFFERENTIALS:
Neutrophils(grans, Adult Absolute: 2-8 x109/L,
polys, PMN, segs) Relative: 45-74 % WBC
Newborn 6-26 x109/
Neutrophil band Adult Absolute: 0.7-10 x109/L
forms Relative: 3-5 %WBC
Lymphocytes Adult Absolute: 1-4.8 x109/L
Relative: 20-50 % WBC
TEST PATIENT NORMAL RANGE UNIT
Lymphocyte Newborn Absolute: 2-11 X109/L
Monocyte Adult Absolute: 0.2-0.8 X109/L
Relative: 3-10 % WBC
Newborn 0.4-3.1 X109/L
Eosinophil Adult Absolute: 0.04-0.5 X109/L
granulocyte Relative: 1-7 %WBC
Newborn 0.02-0.85 X109l/L
Basophil Adult Absolute: 40-900 X109/L
granulocyte Relative: 0-2 %WBC
Newborn 0.6-10 X109/L
PLATELET COUNT 140-450 X103/µl
INTERPRETATION
Certain disease states are defined by an
absolute increase or decrease in the number
of a particular type of cell in the bloodstream.
For example:
CELL TYPE INCREASE DECREASE
RED CELL(RBC) Erythrocytosis or Anemia or
polycythemia e.g. in erythroblastopenia e.g.
polycythemia vera Due to deficiency states,
blood loss, hemolysis etc
WHITE CELL(WBC) Leukocytosis Leukopenia
a)Granulocytes: Granulocytosis Granulocytopenia or
agranulocytosis
i) Neutrophil Neutrophilia- bacterial, Neutropenia
acute viral infections etc
ii) Eosinophils Eosinophilia- parasitic Eosinopenia
infestation, allergy, asthma
iii) Basophils Basophilia- leukemia, Basopenia
lymphoma
CELL TYPE INCREASE DECREASE
b) Lymphocyte Lymphocytosis - viral Lymphopenia- HIV
infections(glandular fever),
CLL
PLATELETS Thrombocytosis- essential Thrombocytopenia- ITP
thrombocytosis
ALL CELL LINES Pancytopenia- aplastic
anemia
BLOOD TRANSFUSION
OVERVIEW
Dr. Kipkulei
Dept of Hematology and Blood Transfusion
DATE: 18/03/2011
BLOOD GROUPS
Major blood group systems

• ABO
• Rhesus
• Kell
• Lewis
• Duffy
• Kidd
• etc
BLOOD GROUPS (cont.)
1. ABO
• Include A, B, AB, O
• Group is determined by presence of relevant
antigens on the RBC surface
• The antigens are glycoproteins and glycolipids
• Antibodies to these antigens may be found in
serum of persons who lack the corresponding
antigens
• The naturally occurring antibodies are usually
IgM
BLOOD GROUPS(cont.)
2. RHESUS(Rh)BLOOD GROUP SYSTEM

• Determined by Cc,D, Ee antigens on RBC
surface
• The D antigen is of major clinical importance
• Anti rhesus antibodies are IgG and arise only
after immunization by transfusion or
pregnancy
NB: The ABO and Rh blood group systems are of
major clinical importance than the other
systems
DEFINITION OF BLOOD TRANSFUSION
• The process of transferring blood or blood

based products from one person(donor) into
the circulatory system of another(recipient)
BLOOD AND BLOOD COMPONENTS FOR
TRANSFUSION
1) Whole blood(420mls of donor blood+120ml

of anticoagulant solution(CPD-A)
2) Red cells(packed red cells)
3) Platelet concentrates
4) Plasma components- whole FFP(contains all
coagulation factors), cryoprecipate(factor
VIII and fibrinogen), specific coagulation
factor concentrate
5) White blood cells- granulocytes
PROCCESS OF OBTAINING BLOOD AND BLOOD
COMPONENTS
Blood donation
• Can be homologous(allogeneic) or autologous
• Donors may be voluntary non-remunerated,
remunerated or patient-recruited/family
replacement
Donor selection
• Must be done by a qualified physician, trained
nurse or clinical officer
• Proper donor selection is important to ensure
blood safety and safety of the donor
Donor selection (cont.)
• Purpose of selection is to identify any factors that
may make an individual as unsuitable as a donor
either temporarily or permanently
• Selection is based on a medical history and a limited
physical exam done on the day of donation.
Criteria for suitable donors
1. Should have no history of risk behaviour
2. Should be health
3. Should give an informed consent
Criteria for suitable donors(cont.)
4. Should have minimal acceptable hemoglobin
of 12.5g/dl (for allogeneic) and 11g/dl(for
autologous
5. Should be between 16 and 65 year
• NB: no more than 10.5ml of whole blood/kg
body weight shall be collected at a donation
Adverse donor reactions
• Fainting or vasovagal syndrome
• Nausea and vomiting
• Twitching or muscular spasms- hyperventilation
• Hematoma formation
• Convulsions
• Cardiac arrest
STORAGE OF COLLECTED BLOOD
• Blood collected during donor campaigns is
stored prior to subjecting to screening tests
• The whole blood is stored at 40C except for
blood meant for platelet preparation, which is
stored at 220centigrade
• Fresh frozen plasma and croprecipitate- stored
at -180C or below
• Blood units collected in CPD-A can be stored
for up to 35 days
PRETRANSFUSION BLOOD TESTS
• Each donor(allogeneic) unit must be tested prior to

release for cross-match and transfusion
• The following tests are carried out
1. Blood group- ABO and Rh
2. Syphylis
3. Hepatits B virus(HBsAg)
4. Hepatitis C virus(anti-HVC)
5. HIV-I&II
USES OF BLOOD AND BLOOD PRODUCTS
a) Red cells
• RBC transfusion is intended to increase the
delivery of O2 to the tissues
i) Whole blood is indicated in replacement of
acute surgical or obstetric blood loss,
autologous transfusion and exchange
transfusion
ii) Packed cells is used in patients who have
chronic anemia or preoperative patients
with Hb<10g/dl and cannot be corrected by
other means
USES OF BLOOD AND BLOOD PRODUCTS….
a) Red cells(cont.)
• A unit of whole blood has a volume of ~ 400-
500ml with Hct of 45-55%
• A unit of PRBCs has ~180-200ml of RBCs and
50-70mls of plasma
• Each unit of blood has ~ 60g of Hb and
250mg of iron
USES OF BLOOD AND BLOOD PRODUCTS…..
a) Red cells(cont.)
• One unit of blood(or the equivalent volume
in a child) usually increases the patient’s Hb
by 1gm/dl
• RBCs are rarely transfused if Hb is >10gm/dl,
usually needed if Hb is < 5gm/dl and may be
needed id Hb is 5-10gm/dl depending on
clinical condintion of the patient
USES OF BLOOD AND BLOOD PRODUCTS…
a) Red cells(cont.)
• In patients with acute blood loss, RBCs
transfusion is needed if tachycardia and
hypotension are not corrected by volume
expanders
USES OF BLOOD AND BLOOD PRODUCTS ….
b) Platelet concentrate
• Indicated in severe thrombocytopenia
(platelets <20×109/l ) and in surgical and
obstetric patients with microvascular
bleeding with platelet count<50,000/mm3
• Is obtained by centrifugation of plasma
• Only platelets of blood group O or A are
routinely collected.
USES OF BLOOD AND BLOOD PRODUCTS ….
b) Platelet concentrate(cont.)
• Cross-matching is not required, but ABO
compatibility should be ensured
• Stored platelets are viable for 3-7 days in
room temperature
• Each unit contains > 5.5×1010 platelets
• Each unit of platelet concentrate increases
the platelet count on an average adult by 7-
10,000/mm3
• The adult therapeutic dose is 4-8 units per
day
c) Plasma products
i. FFP
• is indicated for treatment of bleeding due to
multiple coagulation factor deficiencies, DIC,
massive transfusion with coagulation
abnormalities, and bleeding due to
warfarin(not respondig to vitamin K)
• FFP is frozen within hours of donation
• FFP must be ABO compatible with the
recipient's RBCs
c) Plasma products(cont.)
ii) Cryoprecipitate- indicated in von Willebrand’s
disease, Hemophilia A, factor XIII def,
hypofibrinogenemia
iii) Specific coagulation factors- indicated in
specific coagulation factor deficiency
TYPES OF BLOOD TRANSFUSION
• Homologous(allogeneic) transfusion- transfusion using

the stored blood of other donors
• Autologous transfusion- transfusion using the patient’s
own stored blood
– Perfomed in patients who require elective surgery with Hb
≥ 10gm/dl
– There are 3 ways of administering autologous transfusion:
• Prediposit
• Haemodilution
• Salvage
– For prediposit, collecion should be at least 7 days apart
and last donation should be at least 4 days before surgery
COMPLICATIONS OF BLOOD TRANSFUSION
• Though blood transfusion is a life-saving

therapy, it can result in adverse effects
• Approximaley 1% of all transfusions lead to
some type of adverse reaction
• Physicians must weigh the benefits of
transfusion against the risks
COMPLICATIONS OF BLOOD TRANSFUSION ….
Immediate /early complications

1. Hemolytic transfusion reactions
• Usually due to transfusion of incompatible blood
for the ABO or Rh groups and main problem is
clerical error
• Usually intravascular
• Signs and symptoms include fever chills, nausea,
vomiting, headache, chest pain, dyspnoea,
hypotension, tachycardia, oliguria/anuria, anemia,
DIC, etc
COMPLICATIONS OF BLOOD TRANSFUSION ….
Immediate /early complications….

2. Allergic reactions
• Are common and often due to antigens in
transfused WBCs(commonly HLA) or plasma
proteins
• Include febrile reactions, urticaria, angio-edema,
anaphylactic shock
3. Circulatory overload/fluid overload- causing
acute pulmonary edema
4. Air embolism-less common
COMPLICATIONS OF BLOOD TRANSFUSION(cont)
Immediate /early complications….

5. Massive transfusion- transfusion equivalent of
circulating blood volume or more within 24hrs
• May cause hypocalcemia, hyperkalemia,
coagulopathy due to dilution of platelets and coag
factors, hypothermia, acid-base disturbances
• Measures to reduce problems associated with
massive transfusion include prophylactic use of
FFP, platelet concentrate and use of fresh blood
COMPLICATIONS OF BLOOD TRANSFUSION(cont.)
Delayed/late complications
1. Delayed hemolytic reactions- usually
extravascular, caused by antibodies to non-
D antigen, Kell, Kidd or Duffy antigens
2. Transmission of infections- hepatitis B&C,
HIV, CMV, syphilis, malaria etc
3. Iron overload
• Results from long term transfusion in
patients with aplastic anemia, sickle cell
anemia or thalassaemia
COMPLICATION OF BLOOD TRANSFUSION(cont)
Delayed/late comlications…..
3. Iron overload….
• Deposition of iron in myocardium, liver and endocrine
organs lead to life-threatening clinical problems
• Chelation therapy with desferrioxamine in patients at
risk should be considered
4. Post-Transfusion purpura- develops due to
thrombocytopenia resulting from antiplatelet
antibodies
5. Graft Vs Host disease
MANAGEMENT OF BLOOD TRANSFUSION REACTION
• Stop the blood transfusion

• Start first aid measures- ABC
• Administer hydrocorticosone and antihistamine
• Inform the attending physician
• Monitor the vital signs on the patient
• Inform the laboratory about the event
• Take the following blood samples: 5 mls into a
plain tube and 2 ml into EDTA tube
MANAGEMENT OF BLOOD TRANSFUSION REACTION….
• Send to the lab all samples correctly labeled,

the blood that reacted, with transfusion set,
lab request form
• Report the event to the hospital transfusion
committee
RATIONALE USE BLOOD AND BLOOD PRODUCTS
• Blood should be transfused only when

required to save life
• Stabilize patients with acute blood loss first by
prompt and appropriate supportive care
• Re-evaluate patient immediately prior to
blood transfusion to ensure that transfusion is
still required
RATIONALE USE BLOOD AND BLOOD PRODUCTS….
• Effective transfusion requires a minimum of 2

units for an adult or 20ml of whole blood(10-
15ml of PRBCs) per kg for a child
• Check Hb after transfusion to be done to
assess the efficacy of transfusion
• Blood transfusion is not a cure of anemia and
hence underlying cause should be investigated
‘BLOOD SUBSTITUTES’
VOLUME EXPANDERS
– Crystalloids(normal saline, Ringer’s lactate etc)
– Colloids(dextran, starch, albumin etc)
OXYGEN CARRYING THERAPEUTICS(‘ARTIFICIAL
BLOOD’)
– Perfluorocarbon based
– Hemoglobin based
STEM CELL TRANSPLANTATION
• Indications
• Potential stem cell donors
• Process of obtaining stem cells- conditioning
and collection
• Stem cell processing
• Autologous Vs allogeneic stem cell
transplantation
• Complications
Thank you
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GENERAL PRINCIPLES OF

1) Excessive blood loss

Dr. Kipkulei C.J

• Iron is required for the synthesis of

 Anemia that occurs in patients with:

 Impaired marrow response to anemia –

Dr. Kipkulei J.C

Vitamin B12 deficiency

Dr. Kipkulei C.J

RBC Components that Determine

NB: Free hemoglobin(hemoglobinemia),

βo Means absent gene

• Severe microcytic hypochromic anemia

Relative vs Absolute values

• Neutropenia is an absolute reduction in the

• The blood contain only few percent of total body

• Non-malignant causes of lymphocytosis

• Absolute monocyte count is age dependent

• The causes of eosinophilia can be considered under

• Basophils are least common of the granulocytes

• L1 – Small cells with homogeneous chromatin,

Myeoperoxidase Myelomonocytic cells

Chloroacetate esterase Granulocyte blasts

Alpha naphtybutyrate esterase Monocytic cells

Periodic acid- Schift Lymphoblast, erythroblasts -

• Bone marrow is almost

• Two major categories

Chronic myelogenous leukemia(CML) Chronic Myelogenous Leukemia

Polycythemia Vera(PV) Polycythemia Vera

Essential Thrombocythemia(ET) Essential Thrombocytosis

Agnogenic myeloid Chronic idiopathic myelofibrosis

– A prolonged APTT may be caused by:

• NORMAL 150,000 - 400,000 CELLS/MM 3

• < 150,000 Thrombocytopenia

• 50,000 - 150,000 Mild Thrombocytopenia

• <20,000 severe thrombocytopenia

• Used to test for platelet function

• The INR is calculated from the following

• Prolongation of the aPTT can occur with a

• Heparin is an indirect thrombin inhibitor which

• The thrombin time (TT) measures the final

• Plasmin cleaves fibrin at multiple sites and

• Arterial thromboembolic disease

• Used to assess deficiency of factor XIII

Dr. J.C. Kipkulei

male 13.6-17.2 g/dl

Manifests orally as Pallor of the oral mucosae

• Yellow appearance of the skin, mucous

• Reduced circulating neutrophils [below

• Represents malignancies of hematopoietic stem

Dr. J.C. Kipkulei

– Mean corpuscular hemoglobin

TEST PATIENT NORMAL UNIT COMMENT

Dept of Hematology and Blood Transfusion

Major blood group systems

2. RHESUS(Rh)BLOOD GROUP SYSTEM

• The process of transferring blood or blood

1) Whole blood(420mls of donor blood+120ml

• Each donor(allogeneic) unit must be tested prior to