Haematopathology 3:

Leucocytosis/Leucopenia,
 Peripheral Blood Cells

A. Erythrocytes; B. Large Granular Lymphocyte; C. Neutrophil; D. Eosinophil;

E. Neutrophil; F. Monocyte; G. Platelets; H. Lymphocyte; I. Band Neutrophil; J. B
 White Blood Cell Count

 WBC differential - % of each cell

type in peripheral blood
 Absolute count - more important

% cell x total WBC count

 Reference ranges - based on age,

sex and ethnic background

 White Blood Cell Count
WBC: 4.3 - 11.3 x10 9/L
% Absolute Reference
Counts Range
Neutrophils 65 4550 1,500-7,500
Lymphocyte 30 2100 1,000-4,000
Monocytes 3 210 0 – 800
Eosinophils 1 70 0 – 400
Basophils 1 70 0 - 50
Quantitative Disorders of WBCs
 Leukocytosis -  WBC count
Granulocytosis
Lymphocytosis
Monocytosis
Eosinophilia, Basophilia
 Leukopenia -  WBC count
Neutropenia, Lymphopenia
 Leukocytosis (High WBC)
• Neutrophilia : Increase in the count of neutrophil
• Move from the marginating or storage pool into the circulating
peripheral blood. Next bone marow reserves are shifted into the
peripheral blood and finally marrow production is increased.
 Leukocytosis (High WBC)
Granulocytosis (neutrophilia)
 Acute inflammation

bacterial infections
tissue necrosis
 Leukaemoid reaction -  WBC

immature forms
 Activation - toxic granulation,

dohle bodies
 Conditions related to leukocytosis

• Three condition leukostasis, and

leukemoid and leukoerythroblastic
reactions.
• Leukostasis : results from sludging of
high numbers of leukocytes in small
vessels, particularly the brain, lungs, and
kidneys.
• Leukemoid reactions: characterized by
blasts, promyelocytes, myelocytes, and
metamyelocytes in the peripheral blood.
• May besecondary to benign or malignant
conditions.
• A leukoerythroblastic reaction: is
similar to a leukomoid reaction with the
addition of nucleated red blood cells. A
leukoerythroblastic picture indicates severe
disruption of the marrow
Leukemoid reaction (toxic granulation)
Activated neutrophil - Dohle body
 Leucocytosis (High WBC)
Lymphocytosis
 Most often viral infection

 Chronic inflammation

 “Activated forms” - large

 Marked lymphocytosis with

activated lymphocytes - infectious

mononucleosis (EBV)
Lymphocytes - Normal
Reactive Lymphocyte - Viral Infection
Leukocytosis (High WBC)

Monocytosis
 Chronic infections -
bacterial endocarditis
malaria, tuberculosis
 Chronic inflammation
collagen vascular diseases
Monocytes with vacuoles
 Leukocytosis (High WBC)
Eosinophilia
 Allergic disorders -

hay fever, asthma

drug reactions, skin disease
parasitic infections
Basophilia
 Myeloproliferative disorders
 Leukopenia (Low WBC )
Its due to deficiency of any or all of WBC
Common cause is :
 Neutropenia (Granulocytopenia):

 Is neutrophils of less than 1.5x 10 9/L if < 1 compromise

ability to fight if < 0.5 severe type
 Its clinically significant
• Predisposes to bacterial infections which is
Severe often drug related
• Treat with GM - CSF, G - CSF
 Causes of leucopenia

• Decreased Marrow production:

a)Drug induced b) Aplastic anemia
c) Myelofibrosis d) Ineffective hematopoiesis
• Decreased neutrophil survival (Utilization or destruction)

a)Infection b)Drug induced

c) aggregation d) Hyperspleenism
• Increased margination or storage
• Its typically transient neutropenia with
normal total number of neutrophils and
neutrophil kinetics:
• a) hemodynamic changes
b) viremia
c) hypersensitivity
d) hemolysis
 Minor causes of neutropenia
• Cyclic neutropenia :
• Is an autosomal dominant
• unknown etiology , 3-6 days of neutropenia
• occur every 21-30 days in a periodic Pattern.
• During the periods of neutropenia the patient may
develop fever and infections such as stomatitis, cellulitis,
and vaginitis.
• Chronic idiopathic neutropenia
• Neutrophil count is less than 1.0 x 109/L, but results in
few infections.
•  
 Other forms of leukopenia
• Eosinopenia :( < 0.04 x10 9 /L ) :
• Acute stress (glucocorticoid or epinephrine
stimulation), acute inflammation (interleukin-5),
Cushing's syndrome or corticosteroid
administration.
• Basopenia: ( < 0.01 x10 9 /L )
• Corticosteroid administration, stress, acute
inflammation, and hyperthyroidism.
• Monocytopenia ( < 0.2 x10 9 /L ) after giving
predisone, but by 12 hours returns .
• Lymphocytopenia ( < 1.5 x10 9 /L for adults; <
3.0 x10 9 /L for children )
• Corticosteroid Rx, chemoRx, irradiation,
Hodgkin's disease, HIVinfection, and chronic
diseases ie. sarcoid, TB, lupus.
• Both eosinophils and basophils, like neutrophils,
exhibit diurnal variation, being lowest in the
morning and highest in the evening
• A 35-year-old woman, with a history of depression x1 month was
treated with phenothiazine by Dr. She now presents with a 10 day
history of fever, chills, and malaise. In the past 3 days she noted
increased weakness and fatigability. The chest X-ray shows
evidence of early pneumonia. There is no splenomegaly. Laboratory
values are shown at belwo. The PB smear showed normocytic/
normochromic RBCs.
• What is your diagnosis?
• What is the most likely cause?
• A. ineffective erythropoiesis
B. accelerated destruction of neutrophils
C. drug induced
D. shift from circulating pool to marginal granulocyte pool
E. aplastic anemia
Haematopathology 3:

Malignant WBC disorders

Haematopathology 3:

Malignant disorders
Acute Leukemia, &
Myeloproliferative Disorders
ACUTE LEUKEMIA
Normal Marrow Leukemic Marrow

The marrow is infiltrated by proliferation of immature cells or

blasts. If untreated, death usually occurs within 6 months in most
patients.
 Epidemiology/Etiology
•  
Viral Adult T cell Leukemia/lymphoma
associated with HTLV-I
• African Burkitt's lymphoma/leukemia is
associated with the Epstein-Barr virus.
• Environmental factors: irradiation e.g. atomic
bomb explosions in Japan
• Chemical toxins : e.g. benzene
• Drugs : e.g. chloramphenical, phenylbutazone,
and especially alkalating agent chemotherapy
 Acute Leukemia
• Malignant Transformation of
Haematopoietic Stem Cells
– Acute Lymphoblastic Leukemia
– Acute Myelogenous Leukemia
LEUKEMIA
 Leukaemias
Haematopoietic Precursors

Transformation
Proliferation and Accumulation

Peripheral blood Blasts

Visceral organs Cytopenias
Bone Marrow - Normal (low power)
Bone Marrow - Acute Leukemia (low power)
Bone Marrow - Normal (high power)
Bone Marrow - acute leukemia (high power)
Acute Leukemia - Clinical
Features
• Abrupt Onset
• Loss of normal marrow function
– Anemia, fatigue, fever, infection,
bruising, bleeding
• Infiltration of organs
– Hepatomegaly, splenomegaly
• CNS manifestations
– Headache, nausea, nerve palsy
 Acute Leukemia

 Untreated patients survive <6 months

 Age predominance
ALL - children (peak incidence 4 yrs)
AML - adults (median age 50 yrs)
FAB classification (1975;1985 revision)
 ACUTE
LYMPHOBLASTIC
LEUKEMIA (ALL)
 Acute Lymphoblastic Leukemia
 Children and young adults
 20 fold increase incidence of leukemia in
children with Down's syndrome.
 Diagnosis - >20% lymphoblasts in BM
- cytochemical stains - TdT +, MPO -
- flow cytometry - cell surface markers

B cells T cells

 Classified into subgroups based on cell surface

markers and cytogenetics
Subclassification

• ALL
– Common type( pre-B)
– B-cell
– T-cell
– Undifferentiated
 laboratory evaluation
• CBC: including a platelet count and an
examination of a peripheral blood smear.
• WBCc: is typically increasedWBCc is really
highly variable, ranging from severe leukopenia
to extreme leukocytosis.
• platelet count: is decreased
• Hct and Hb : normocytic/normochromic anemia
• PBP: Blasts or immature cells. Occasionally,
patients present with no peripheral blasts
(aleukemic leukemia)
 Blast Morphology

Lymphoblasts Myeloblasts
Similarities round-oval nuclei round-oval nuclei
Differences

Nuclear coarse chromatin which fine delicate chromatin

tends to aggregate into
masses
Cytoplasmic more basophilic no less basophilic has
azurophilic granules no primary azurophilic
Auer rods may have granules may have Auer
non-specific rods
cytoplasmic granules
Lymphoblasts - Acute Lymphoblastic
Leukemia
TdT positive lymphoblasts (green fluorescence)
 Acute Lymphoblastic Leukemia
 Responsive to therapy
Good Prognosis
- majority “cured” with chemotherapy
 Age 2-10 yrs

 Female

 Early B - precursor phenotype

 Hyperdiploidy (>50 chromosomes/cell)

 WBC count <10,000

 ACUTE
MYELOGENOUS
LEUKEMIA (AML)
 Acute Myelogenous Leukemia

 Adults (20% of childhood leukemia)

 Responds poorly to chemotherapy

- 15-30% long term DFS

- allogeneic BMT curative
 FAB Classification for AML
(French-American-British Classification)
 Published 1976
 AML if >30% blasts in BM
 8 groups (cell type and degree of

differentiation)
Morphology
Cytochemistry
FAB classification
 Clinical features
General :
Onset is abrupt & stormy
(usually present within 3 months)

– Bone marrow failure

(anemia, infection ,bleeding)

– Bone pain & tenderness

Specific:
• M2 : Chloroma:-presents as a mass lesion
‘tumor of leukemic cells’
• M3 : DIC
• M4/M5 : Infiltration of soft tissues,
gum infiltration,
infiltration skin deposits
,Meningeal involvement-headache, vomiting,
eye symptoms
Skin Infiltration with AML (Leukemia Cutis)
 Diagnosis
• Blood count :
WBC usually elevated (50,000- 1,00,000
/ cmm ); may be normal or low;
often anemia & thrombocytopenia

• Blood film : (as above)

Blast cells
P. Smear AML
• Bone marrow aspirate & trephine:
Hypercellular,
– blast cells ( > 20%),
– presence of Auer rods - AML type

• Cytochemistry :
Special stains to differentiate AML
from ALL ;
Positivity with Sudan black &
Myeloperoxidase (MPO) in AML
Myeloblasts - myeloproxidase positive
MPO (right) & Sudan black (left)
showing intense localised positivity in
blasts
 Myeloblasts - AML

er ro d
Au
Auer Rods in Leukemia cells
• Confirmation:
– Immunophenotyping
– Molecular genetics
– Cytogenetics: Chromosomal abnormalities
 FAB - M3
Acute Promyelocytic Leukemia
 Hypergranular promyelocytes
 Increased Auer rods
 DIC from tissue thromboplastin
 Give retinoic acid
 15;17 chromosomal translocation
(retinoic acid receptor gene)
Acute promyelocytic leukemia - AML M3
 AML - Molecular Biology

Retinoic Acid Receptor Gene

 Gene on chromosome 17
 Regulates cell differentiation
Monoblasts - AML (M5)
MYELOPROLIFERATIVE
DISORDERS
 MPD
• Is malignant clonal proliferations of
multipotent stem cells.
• All three cell lines (myeloid, erythroid
and megakaryocytic) are involved in
each disorder
• Each has specific genetic abnormalities
• The dominant cell differs in each
allowing for the subclassification of the
chronic myeloproliferative disorders
 Chronic myeloproliferative
disorder

1) acquired, malignant clonal disorders

2) characterized by expansion of pluripotent
stem cells
3) deranged production of one or more
myeloid lines
4) a variable predisposition to transform to
leukemia.
 Myeloproliferative Disorders

 Middle aged - elderly individual

 Chronic process - more indolent
than acute leukemia
Classification of MPD
 Myeloproliferative Disorders
Clinical manifestations
 Splenomegaly

 Cellular phase -

hypercellular BM
peripheral blood cytoses
 Fibrotic and/or leukemic phase -

progressive BM fibrosis
AML or ALL (20%)
 Myeloproliferative Disorders

 Chronic Myeloid Leukemia - granulocytes

 Polycythaemia Vera - red cells
 Myelofibrosis with Myeloid Metaplasia -
fibrosis
 Essential Thrombocythaemia - platelets
 Chronic Myeloid Leukemia
(CML)
 WBC from 20,000 to > 100,000
 WBC > 50,000 (80%) with immature

myeloid cells, basophils, eosinophils

 Unique chromosomal abnormality -

Philadelphia chromosome

Slowly progressive  accelerated
  phase
80% blast phase (ALL or AML)
 Clinical Course

• The initial phase of CML is stable or

indolent (usually lasting 2-4 years)
• An accelerated stage (6-12 months)
• An acute phase or blast crisis (2-4
months) similar to acute leukemia.
Chronic Myeloid Leukemia - Peripheral blood smear
Chronic Myeloid Leukemia - Bone marrow biopsy
Philadelphia Chromosome (Ph)
 Reciprocal translocation t(9;22)
 Results in bcr/abl gene fusion

c-abl (Abelson) chromosome 9

bcr (break point cluster region)
chromosome 22
 Protein with tyrosine kinase activity

- plays critical role in pathogenesis

Chronic Myeloid Leukemia - t(9;22) chromosomal
translocation
(Philadelphia chromosome)
 Polycythaemia Vera (PV)
Increased erythrocyte cell mass

Increased
Haematocrit
Blood volume
Blood viscosity

Thrombotic or hemorrhagic problems

 Differential Diagnosis

• 1) Relative increases in red cell concentration

(hemoconcentration,ie. dehydration)
• 2) Secondary forms of absolute increases in red
cell number (increased erythropoietin, ie.
smoking & sleep apnea).
 Diagnostic criteria

A B
A1 -increased RBC mass B1 -thrombocytosis
>36ml/kg in men >400 x10 /L
>32ml/kg in women
A2 - normal arterial O2 B2 - leukocytosis (no fever/infection)
>12 x10 /L

A3 – splenomegaly B3-
increased LAP >100
increased B12 >900pgml

PV is diagnosed if all column "A" criteria are met or if A1

and A2 plus any two from column "B"
 PV - Natural History
 Indolent
 Treat symptoms (i.e. phlebotomy)
 < 25% progress
Marked BM fibrosis (spent
phase)
Acute leukemia (2-15%)
Myeloid metaplasia with myelofibrosis - Bone marrow fibrosis
 Myeloid Metaplasia with
Myelofibrosis (MMM)
Originally agnogenic myeloid metaplasia

etiology extramedullary
unknown (  spleen)

 Abnormal stem cells  deranged

haematopoietic cells (esp. megakaryocytes)
 stimulate fibroblasts
 MMM - Natural History
Initial cellular phase
Progressive BM fibrosis / failure

Infection
Hemorrhage
Acute leukemia
(< 10%)
• PBP: The platelet count often exceeds one
million/mL
• Must be carefully differentiated from reactive
thrombocytoses various malignancies, iron
deficiency anemia, etc.
• The other chronic myeloproliferative disorders
CML, PV and 1'MF must be ruled out.
• Bone Marrow: hypercellular with excessive
numbers of megakaryocytes and can be difficult to
distinguish from polycythemia vera.
Essential Thrombocythemia - Bone marrow with greatly
increased numbers of megakaryocytes
 Essential Thrombocythaemia
 platelets - episodic symptoms

Bleeding /
thrombosis

Unrelated

Acute leukemia
(< 1%)
 Diagnostic Criteria
• Platelets >600 x10 /L
• Hemoglobin <130g/L
• Marrow iron stores or failed Fe trial
• Philadelphia chromosome negative
• No collagenous fibrosis of marrow or fibrosis
<1/3 examined area with no splenomegaly
and no leukoerythroblastic reaction
• No known cause of reactive thrombocytosis