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
Neutrophil
Platelets

Eosinophil
Small Erythrocyte
lymphocyte

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—antigen-
presenting 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 Colony­forming Precursor Mature
stem cell units (CFUs) cells cells

Eosinophilic Eosinophilic Eosinophilic Eosinophilic leaves

Eosinophil
CFU myeloblast promyelocyte myelocyte

Basophilic Basophilic Basophilic Basophilic Basophil

CFU myeloblast promyelocyte myelocyte

Neutrophilic Neutrophilic Neutrophilic Neutrophilic Neutrophil

CFU myeloblast promyelocyte myelocyte

Monocytic Monoblast Promonocyte Monocyte

CFU

B lymphocyte
B prolymphocyte

T prolymphocyte T lymphocyte
Lymphocytic Lymphoblast
CFU

18-13 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
ALL=Acute  others
11%
Lymphocytic 17%
CML=Chronic 
Mylogenous
AML=Acute 
Mylogenous CML CLL
15% 26%

AML
31%

Total Reported Cases = 31,500

 Pictures Of Blood
Platelet Platelet
White Cell 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 Stage 5a- Anemia

 Red Cell
 Platelet Stage 4- Worsening
 Blast
 Germ
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
 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
 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
 Myeloperoxidas
e

(MPO)

p­Phenylene diamine + Catecol + H2O2

        MPO      
> Brown black deposits

Brown deposits considered to be a positive test­
differentiates AML from other leukemias
M1 and M2
M4 M3

M5
Chloracetate (Specific) Esterase

Myeloid Cell Line

Naphthol­ASD­chloracetate
    CAE     
> Free naphthol compounds
+ Stable diazonium salt (eg, Fast Corinth)
                  
> Red deposit
 Non­Specific 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  WHO criteria
 Morphology
 Morphology
 Immunophenotyping
 Cytochemistry
 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)
 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
      (para­rosaniline, 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 Hypoploidy
Trisomy 4, 10, 17 Trisomy 5
t(12;21) (TEL/AML1) t(1;19 (E2A/PBX1)
Mixed lineage leukemia
T(9;22) (Ph)

BM blast count Mkd reduction at day 7 Mild reduction at day 7

during induction
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 AS­MX phosphate    LAP at pH8.6  >
Naphthol AS­MX + 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 1+

2+ 3+ 4+
LAP Score
Example:

0 x 35 cells =    0
1+ x 30 cells =  30
2+ x 20 cells =  40
3+ x 10 cells =  30
4+ x 5 cells =  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