Leukemia/Lymphoma
Components and General Properties
of Blood

Seven kinds of formed elements


Erythrocytes: red blood cells (RBCs)
Platelets (thrombocytes) (clotting cells)


Cell fragments from special cell in bone marrow
Leukocytes: white blood cells (WBCs)-5 kinds
18-2
Components and General Properties
of Blood

Five leukocyte types divided into two categories
 Granulocytes (with granules)
• 3)Neutrophils
• 4)Eosinophils
• 5)Basophils

Agranulocytes (without granules)
• 6)Lymphocytes
• 7)Monocytes
Components and General Properties
of Blood
Monocyyte
Small
lymphocyte
Platelets
Neutrophil
Eosinophil
Small
lymphocyte
Erythrocyte
Young (band)
neutrophil
Neutrophil
Monocyte
Large
lymphocyte
Neutrophil
Basophil
Leukocytes

Least abundant formed element
– 5,000 to 10,000 WBCs/L





Protect against infectious microorganisms and other
pathogens
Conspicuous nucleus
Spend only a few hours in the bloodstream before migrating
to connective tissue
Retain their organelles for protein synthesis
Granules


All WBCs have lysosomes called nonspecific (azurophilic) granules:
inconspicuous so cytoplasm looks clear
Granulocytes have specific granules that contain enzymes and other
chemicals employed in defense against pathogens
18-5
Types of Leukocytes

Granulocytes
 Neutrophils (60% to 70%): polymorphonuclear leukocytes
 Barely visible granules in cytoplasm; three- to five-lobed
nucleus
 Eosinophils (2% to 4%)
 Large red-orange granules;
 Basophils (less than 1%)
 Large, abundant, violet granules
18-6

Agranulocytes

Lymphocytes (25% to 33%)


Variable amounts of bluish cytoplasm (scanty to abundant);
ovoid/round, uniform dark violet nucleus
Monocytes (3% to 8%)

Largest WBC; generally ovoid, kidney-, or horseshoe-shaped
nucleus
Granulocytes

Neutrophils—increased numbers in bacterial infections
 Phagocytosis of bacteria
 Release antimicrobial chemicals

Eosinophils—increased numbers in parasitic infections, collagen
diseases, allergies, diseases of spleen and CNS
 Phagocytosis of antigen–antibody complexes,
allergens, and inflammatory chemicals
 Release enzymes to destroy large parasites
18-8

Basophils—increased numbers in chickenpox, sinusitis,
diabetes


Secrete histamine (vasodilator): speeds flow of blood to an injured
area
Secrete heparin (anticoagulant): promotes the mobility of other
WBCs in the area
Agranulocytes

Lymphocytes—increased numbers in diverse
infections and immune responses




Destroy cells (cancer, foreign, and virally infected cells)
“Present” antigens to activate other immune cells
Coordinate actions of other immune cells
Secrete antibodies and provide immune memory
18-10
Agranulocytes

Monocytes—increased numbers in viral infections
and inflammation

Leave bloodstream and transform into macrophages


Phagocytize pathogens and debris
“Present” antigens to activate other immune cells—antigenpresenting cells (APCs)
18-11
The Leukocyte Life Cycle

Leukopoiesis—production of white blood cells

Pluripotent stem cells (PPSCs)
Myeloblasts—form neutrophils,
eosinophils, basophils
 Monoblasts—form monocytes
 Lymphoblasts give rise to all forms of
lymphocytes

18-12
Leukopoiesis
Pluripotent
stem cell
Colony-forming
units (CFUs)
Mature
cells
Precursor
cells
Eosinophilic
myeloblast
Eosinophilic
promyelocyte
Eosinophilic
myelocyte
Eosinophil
Basophilic
CFU
Basophilic
myeloblast
Basophilic
promyelocyte
Basophilic
myelocyte
Basophil
Neutrophilic
CFU
Neutrophilic
myeloblast
Neutrophilic
promyelocyte
Neutrophilic
myelocyte
Neutrophil
Monocytic
CFU
Monoblast
Promonocyte
B prolymphocyte
Lymphocytic
CFU
18-13
leaves
Eosinophilic
CFU
Lymphoblast
Monocyte
B lymphocyte
T prolymphocyte
T lymphocyte
NK prolymphocyte
NK cell
What Is Leukemia?
Cancer of the white blood cells
 Acute or Chronic
 Affects ability to produce normal blood
cells
 Bone marrow makes abnormally large
number of immature white blood cells
called blasts

History

Means “white blood” in Greek

Discovered by Dr. Alfred Velpeau in
France, 1827

Named by pathologist Rudolf Virchow in
Germany, 1845
Leukocyte Disorders

Leukemia—cancer of hemopoietic tissue that usually
produces an extraordinary high number of circulating
leukocytes and their precursors


Myeloid leukemia: uncontrolled granulocyte production
Lymphoid leukemia: uncontrolled lymphocyte or monocyte
production
18-16
Acute vs Chronic Leukemia
Acute leukemia: appears suddenly, progresses
rapidly, death within months –blasts found in
peripheral blood
 Chronic leukemia: undetected for months,
survival time average of 3 years
 Effects: normal cell percentages disrupted;
impaired clotting; opportunistic infections

Main Types

Acute Lymphocytic Leukemia (ALL)

Acute Myelogenous Leukemia (AML)

Chronic Lymphocytic Leukemia (CLL)

Chronic Myelogenous Leukemia (CML)
Demographics of Leukemia
Patients (2001 Data)
CLL=Chronic
Lymphocytic
ALL=Acute
Lymphocytic
ALL
11%
others
17%
CML=Chronic
Mylogenous
AML=Acute
Mylogenous
CML
15%
CLL
26%
AML
31%
Total Reported Cases = 31,500
Pictures Of Blood
Platelet
White Cell
Platelet
Red Cell
Red Cell
Blasts
White Cell
Normal human blood
Blood with leukemia
Development of Leukemia in the
Bloodstream
Stage 1- Normal
Stage 2- Symptoms
Stage 3- Diagnosis
Legend
White Cell
Red Cell
Platelet
Blast
Germ
Stage 5a- Anemia
Stage 4- Worsening
Stage 5b- Infection
Causes

High level radiation/toxin exposure

Viruses

Genes

Chemicals

Mostly unknown
Signs and Symptoms of AML
Insidious nonspecific onset
 Pallor due to anemia
 Febrile (fever) due to ineffective WBC
 Petechiae (skin bruising) due to
thrombocytopenia
 Bone pain

Petechiae
Typical Labs
of AML
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

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Leukocytosis
Blastemia
Leukemic hiatus
Auer rods – only found
in myelocytic blasts
Thrombocytopenia
Anemia
>20% blasts in Bone
Marrow
Auer Rods
Auer
Rod
CD Markers
The cluster of differentiation (cluster of
designation) (often abbreviated as CD) is a
protocol used for the identification and
investigation of cell surface molecules
providing targets for immunophenotyping
of cells.
 The CD markers can be used to identify the
type of cell.

Other Findings
CD 13 and CD 33 in flowcytometry
 Cytochemistries-stains that can be used to
differentiate leukemias
 Myeloperoxidase
 Sudan black B
 Choloroacetate esterase (specific)
 Nonspecific esterase

Flow Cytometry
Large clustering of CD 33s shows presence
of blasts
FAB (1976) Classification

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M0 -- Undifferentiated AML
M1 -- AML without maturation
M2 -- AML with maturation
M3 -- Acute Promyelocytic Leukemia
M4 -- Acute Myelomonocytic Leukemia
M5 -- Acute Monocytic Leukemia
M6 -- Erythroleukemia (DiGuglielmo’s)
M7 -- Megakaryoblastic Leukemia
Myeloperoxidase
(MPO)
p-Phenylene diamine + Catecol + H2O2
MPO
> Brown black deposits
Brown deposits considered to be a positive testdifferentiates AML from other leukemias
M1 and M2
M3
M4
M5
Chloracetate (Specific) Esterase
Myeloid Cell Line
Naphthol-ASD-chloracetate
CAE > Free naphthol compounds
+ Stable diazonium salt (eg, Fast Corinth)
> Red deposit
Non-Specific Esterase
Monocytic Line
 Naphthyl acetate
ANAE > Free naphthyl compounds
+Stable diazonium salt (eg, Fast blue RR)
> Brown deposits
FAB vs WHO Classifications of
Hematologic Neoplasm

FAB criteria
 Morphology
 Cytochemistry

WHO criteria
 Morphology
 Immunophenotyping
 Genetic features
 Karyotyping
 Molecular testing
 Clinical features
WHO Classification of AML
AML with recurrent cytogenic
translocations
 AML with multi-lineage dysplasia
 AML and myelodysplasia, therapy related
 AML, not otherwise categorized

AML with Recurrent Cytogenetic
Translocations (WHO 1995)


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t(8;21) -- some maturation of neutrophilic line;
rare in older patients; AML1/ETO fusion protein;
>90% FAB M2
t(15;17) -- APL (granular and microgranular
variants); retinoic acid receptor (RAR) leukemias;
middle aged adults; DIC
inv(16) or t(16;16) -- monocytic and granulocytic;
abnormal eosinophilic component
11q23 -- monocytic; children; most common is
t(9;11)
Lymphocytic Leukemias

Can involve T or B lymphocytes
 B lymphs mature in bone marrow
 Responsible for making antibodies
 T lymphs go to thymus to mature
 Are cytotoxic cells of immune system
FAB Classification of ALL
L1: Small homogeneous blasts; mostly in
children
 L2: Large heterogeneous blasts; mostly in
adults
 L3: “Burkitt” large basophilic B-cell blasts
with vacuoles

L2
L3
Periodic Acid Schiff
Periodic acid + Glycogen
oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na metabisulfite)
> Red deposit
ALL Cytochemistries
Oil Red O: stains L3 vacuoles
 Terminal deoxynucleotidyl transferase
(Tdt): DNA polymerase in early
lymphoblasts
 Cell surface markers (CD’s)
 Cytoplasmic and surface immunoglobulins:
B-cell line
 T-cell receptor (TCR)

WHO Classification of
Lymphoproliferative Syndromes



Precursor B Lymphoblastic Leukemia/Lymphoma
(ALL/LBL) -- ALL in children (80-85% of
childhood ALL); LBL in young adults and rare;
FAB L1 or L2 blast morphology
Precursor T ALL/LBL -- 15% of childhood ALL
and 25% of adult ALL
Burkitt Leukemia/Lymphoma (FAB L3)
Prognosis
Indicators
Favorable
Poor
WBC
< 50,000/L
 50,000/L
Age
1 - 10
< 1 or 10
Gender
Female
Male
Blast
B-cell
T-cell and mixed
Karyotype
Hyperploidy
Trisomy 4, 10, 17
t(12;21) (TEL/AML1)
Hypoploidy
Trisomy 5
t(1;19 (E2A/PBX1)
Mixed lineage leukemia
T(9;22) (Ph)
BM blast count Mkd reduction at day 7
during induction
Mild reduction at day 7
Burkitt’s Lymphoma
CML
Typical Labs in CML
Leukocytosis with blastemia
 Thrombocytosis
 Basophilia
 Micro-megakaryocytes
 Low LAP score (intermediate if infected)
 About 10% blasts in BM
 Philadelphia chromosome

Bone marrow aspirate
and biopsy

Conventionally, a leukocytosis exceeding
50,000 WBC/mm3 with a significant
increase in early neutrophil precursors is
referred to as a leukemoid reaction.

Serum leukocyte alkaline phosphatase is
normal or elevated in leukemoid reaction,
but is depressed in chronic myelogenous
leukemia.

Leukemoid reactions are generally benign
and are not dangerous in and of themselves,
although they are often a response to a
significant disease state

Historically, various clues including the
leukocyte alkaline phosphatase score and
the presence of basophilia were used to
distinguish CML from a leukemoid
reaction. However, at present the test of
choice in adults to distinguish CML is an
assay for the presence of the Philadelphia
chromosome, either via cytogenetics and
FISH, or via PCR for the BCR/ABL fusion
gene.
Leukocyte Alkaline
Phosphatase (LAP)
Naphthol AS-MX phosphate LAP at pH8.6 >
Naphthol AS-MX + Diazonium salt
(eg, Fast blue RR)
> Insoluble pigment
LAP Score
Count 100 consecutive segmented
neutrophils and bands
 Score:
0 = no granules
1+ = occasional diffuse granules
2+ = moderate number of granules
3+ = many strongly positive granules
4+ = confluent strongly positive granules

0
2+
1+
3+
4+
LAP Score
Example:
0
1+
2+
3+
4+
x
x
x
x
x
35 cells
30 cells
20 cells
10 cells
5 cells
= 0
= 30
= 40
= 30
= 20
120 LAP Score
Philadelphia Chromosome





9 ;22 translocation almost specific to CML
Karyotype to visualize Ph chromosome
Produces BCR/c-abl fusion oncogene
Gene product p190 is a hyperactive tyrosine
kinase
Ph chromosome seen in ALL produces p210 and
chronic neutrophilic leukemia produces p230
Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome
FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion
signals (arrow): A=positive (contains a residual ABL signal), B=normal
Chronic Lymphocytic Leukemia
Exclusive in elderly
 Lymphocytosis unrelated to viral infection
 Hyper-mature lymphocytes with highly
condensed nuclei
 Smudge cells

CLL
PB and BM
Smudge cell
Bone Marrow