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Leukemia/Lymphoma
18-2
Components and General Properties of Blood
Seven kinds of formed elementsSeven kinds of formed elements Erythrocytes: red blood cells (RBCs)Erythrocytes: red blood cells (RBCs) Platelets (thrombocytes) (clotting cells)Platelets (thrombocytes) (clotting cells)
Cell fragments from special cell in bone marrowCell fragments from special cell in bone marrow Leukocytes: white blood cells (WBCs)-5 kindsLeukocytes: white blood cells (WBCs)-5 kinds
Components and General Properties of Blood
Five leukocyte types divided into two categoriesFive leukocyte types divided into two categories Granulocytes (with granules)Granulocytes (with granules)
• 3)Neutrophils3)Neutrophils• 4)Eosinophils4)Eosinophils• 5)Basophils5)Basophils
Agranulocytes (without granules)Agranulocytes (without granules)
• 6)Lymphocytes6)Lymphocytes• 7)Monocytes7)Monocytes
Components and General Properties of Blood
Neutrophil
Erythrocyte
Eosinophil
Monocyte
Neutrophil
Basophil
Neutrophil
Platelets
Monocyyte
Smalllymphocyte
Young (band)neutrophil
Smalllymphocyte
Largelymphocyte
18-5
Leukocytes
Least abundant formed elementLeast abundant formed element– 5,000 to 10,000 WBCs/5,000 to 10,000 WBCs/LL
Protect against infectious microorganisms and other Protect against infectious microorganisms and other pathogenspathogens
Conspicuous nucleusConspicuous nucleus Spend only a few hours in the bloodstream before migrating Spend only a few hours in the bloodstream before migrating
to connective tissueto connective tissue Retain their organelles for protein synthesisRetain their organelles for protein synthesis GranulesGranules
All WBCs have lysosomes called nonspecific (azurophilic) granules: All WBCs have lysosomes called nonspecific (azurophilic) granules: inconspicuous so cytoplasm looks clearinconspicuous so cytoplasm looks clear
Granulocytes have specific granules that contain enzymes and other Granulocytes have specific granules that contain enzymes and other chemicals employed in defense against pathogenschemicals employed in defense against pathogens
18-6
Types of Leukocytes
GranulocytesGranulocytes Neutrophils (60% to 70%): polymorphonuclear leukocytesNeutrophils (60% to 70%): polymorphonuclear leukocytes
Barely visible granules in cytoplasm; three- to five-lobed Barely visible granules in cytoplasm; three- to five-lobed nucleusnucleus
Eosinophils (2% to 4%) Eosinophils (2% to 4%) Large red-orange granules; Large red-orange granules;
Basophils (less than 1%) Basophils (less than 1%) Large, abundant, violet granulesLarge, abundant, violet granules
AgranulocytesAgranulocytes Lymphocytes (25% to 33%) Lymphocytes (25% to 33%)
Variable amounts of bluish cytoplasm (scanty to abundant); Variable amounts of bluish cytoplasm (scanty to abundant); ovoid/round, uniform dark violet nucleus ovoid/round, uniform dark violet nucleus
Monocytes (3% to 8%) Monocytes (3% to 8%) Largest WBC; generally ovoid, kidney-, or horseshoe-shaped Largest WBC; generally ovoid, kidney-, or horseshoe-shaped
nucleusnucleus
18-8
Granulocytes
Neutrophils—increased numbers in bacterial infectionsNeutrophils—increased numbers in bacterial infections Phagocytosis of bacteriaPhagocytosis of bacteria Release antimicrobial chemicalsRelease antimicrobial chemicals
Eosinophils—increased numbers in parasitic infections, collagen Eosinophils—increased numbers in parasitic infections, collagen diseases, allergies, diseases of spleen and CNSdiseases, allergies, diseases of spleen and CNS Phagocytosis of antigen–antibody complexes, Phagocytosis of antigen–antibody complexes,
allergens, and inflammatory chemicalsallergens, and inflammatory chemicals Release enzymes to destroy large parasitesRelease enzymes to destroy large parasites
Basophils—increased numbers in chickenpox, sinusitis, Basophils—increased numbers in chickenpox, sinusitis, diabetesdiabetes Secrete histamine (vasodilator): speeds flow of blood to an injured Secrete histamine (vasodilator): speeds flow of blood to an injured
areaarea Secrete heparin (anticoagulant): promotes the mobility of other Secrete heparin (anticoagulant): promotes the mobility of other
WBCs in the areaWBCs in the area
18-10
Agranulocytes
Lymphocytes—increased numbers in diverse Lymphocytes—increased numbers in diverse infections and immune responsesinfections and immune responses
Destroy cells (cancer, foreign, and virally infected cells)Destroy cells (cancer, foreign, and virally infected cells) ““Present” antigens to activate other immune cellsPresent” antigens to activate other immune cells Coordinate actions of other immune cellsCoordinate actions of other immune cells Secrete antibodies and provide immune memorySecrete antibodies and provide immune memory
18-11
Agranulocytes
Monocytes—increased numbers in viral infections Monocytes—increased numbers in viral infections and inflammationand inflammation
Leave bloodstream and transform into macrophagesLeave bloodstream and transform into macrophages Phagocytize pathogens and debrisPhagocytize pathogens and debris ““Present” antigens to activate other immune cells—antigen-Present” antigens to activate other immune cells—antigen-
presenting cells (APCs)presenting cells (APCs)
18-12
The Leukocyte Life Cycle
Leukopoiesis—production of white blood cellsLeukopoiesis—production of white blood cells Pluripotent stem cells (PPSCs)Pluripotent stem cells (PPSCs)
Myeloblasts—form neutrophils, Myeloblasts—form neutrophils, eosinophils, basophilseosinophils, basophils
Monoblasts—form monocytesMonoblasts—form monocytesLymphoblasts give rise to all forms of Lymphoblasts give rise to all forms of
lymphocyteslymphocytes
18-13
Leukopoiesis
leaves
Lymphoblast
B prolymphocyteB lymphocyte
T lymphocyte
NK cell
T prolymphocyte
NK prolymphocyte
Monoblast Promonocyte Monocyte
Basophil
Eosinophil
Neutrophil
Pluripotentstem cell
Colony-formingunits (CFUs)
Precursorcells
Maturecells
Eosinophilicmyelocyte
Eosinophilicpromyelocyte
Eosinophilicmyeloblast
EosinophilicCFU
BasophilicCFU
NeutrophilicCFU
MonocyticCFU
LymphocyticCFU
Basophilicmyeloblast
Neutrophilicmyeloblast
Neutrophilicpromyelocyte
Basophilicpromyelocyte
Basophilicmyelocyte
Neutrophilicmyelocyte
What Is Leukemia?
Cancer of the white blood cellsCancer of the white blood cells Acute or ChronicAcute or Chronic Affects ability to produce normal blood Affects ability to produce normal blood
cellscells Bone marrow makes abnormally large Bone marrow makes abnormally large
number of immature white blood cells number of immature white blood cells called blastscalled blasts
History
Means “white blood” in GreekMeans “white blood” in Greek
Discovered by Dr. Alfred Velpeau in Discovered by Dr. Alfred Velpeau in France, 1827France, 1827
Named by pathologist Rudolf Virchow in Named by pathologist Rudolf Virchow in Germany, 1845Germany, 1845
18-16
Leukocyte Disorders
Leukemia—cancer of hemopoietic tissue that usually Leukemia—cancer of hemopoietic tissue that usually produces an extraordinary high number of circulating produces an extraordinary high number of circulating leukocytes and their precursorsleukocytes and their precursors
Myeloid leukemia: uncontrolled granulocyte productionMyeloid leukemia: uncontrolled granulocyte production Lymphoid leukemia: uncontrolled lymphocyte or monocyte Lymphoid leukemia: uncontrolled lymphocyte or monocyte
productionproduction
Acute vs Chronic Leukemia
Acute leukemia: appears suddenly, progresses Acute leukemia: appears suddenly, progresses rapidly, death within months –blasts found in rapidly, death within months –blasts found in peripheral bloodperipheral blood
Chronic leukemia: undetected for months, Chronic leukemia: undetected for months, survival time average of 3 yearssurvival time average of 3 years
Effects: normal cell percentages disrupted; Effects: normal cell percentages disrupted; impaired clotting; opportunistic infectionsimpaired clotting; opportunistic infections
Main Types
Acute Lymphocytic Leukemia (ALL)Acute Lymphocytic Leukemia (ALL)
Acute Myelogenous Leukemia (AML)Acute Myelogenous Leukemia (AML)
Chronic Lymphocytic Leukemia (CLL)Chronic Lymphocytic Leukemia (CLL)
Chronic Myelogenous Leukemia (CML)Chronic Myelogenous Leukemia (CML)
Demographics of Leukemia Patients (2001 Data)
ALL11%
CLL26%
AML31%
CML15%
others17%
Total Reported Cases = 31,500
CLL=Chronic Lymphocytic
ALL=Acute Lymphocytic
CML=Chronic Mylogenous
AML=Acute Mylogenous
Pictures Of Blood
Normal human blood
White Cell Red Cell
Platelet
Blood with leukemia
BlastsRed Cell
Platelet
White Cell
Development of Leukemia in the Bloodstream
Stage 1- Normal Stage 2- Symptoms Stage 3- Diagnosis
Stage 4- Worsening
Stage 5a- Anemia
Stage 5b- Infection
Legend
White Cell
Red Cell Platelet Blast Germ
Causes
High level radiation/toxin exposureHigh level radiation/toxin exposure
VirusesViruses
GenesGenes
ChemicalsChemicals
Mostly unknownMostly unknown
Signs and Symptoms of AML
Insidious nonspecific onsetInsidious nonspecific onset Pallor due to anemiaPallor due to anemia Febrile (fever) due to ineffective WBCFebrile (fever) due to ineffective WBC Petechiae (skin bruising) due to Petechiae (skin bruising) due to
thrombocytopeniathrombocytopenia Bone painBone pain
PetechiaePetechiae
Typical Labs of AML
LeukocytosisLeukocytosis BlastemiaBlastemia Leukemic hiatusLeukemic hiatus Auer rods – only found Auer rods – only found
in myelocytic blastsin myelocytic blasts ThrombocytopeniaThrombocytopenia AnemiaAnemia >20% blasts in Bone >20% blasts in Bone
MarrowMarrow
Auer Rods
Auer Rod
CD Markers
The The cluster of differentiationcluster of differentiation ( (cluster of cluster of designationdesignation) (often abbreviated as ) (often abbreviated as CDCD) is a ) is a protocol used for the identification and protocol used for the identification and investigation of cell surface molecules investigation of cell surface molecules providing targets for providing targets for immunophenotyping of cells. of cells.
The CD markers can be used to identify the The CD markers can be used to identify the type of cell.type of cell.
Other Findings
CD 13 and CD 33 in flowcytometryCD 13 and CD 33 in flowcytometry Cytochemistries-stains that can be used to Cytochemistries-stains that can be used to
differentiate leukemiasdifferentiate leukemias MyeloperoxidaseMyeloperoxidase Sudan black BSudan black B Choloroacetate esterase (specific)Choloroacetate esterase (specific) Nonspecific esteraseNonspecific esterase
Flow Cytometry
Large clustering of CD 33s shows presence of blasts
FAB (1976) Classification
M0 -- Undifferentiated AMLM0 -- Undifferentiated AML M1 -- AML without maturationM1 -- AML without maturation M2 -- AML with maturationM2 -- AML with maturation M3 -- Acute Promyelocytic LeukemiaM3 -- Acute Promyelocytic Leukemia M4 -- Acute Myelomonocytic LeukemiaM4 -- Acute Myelomonocytic Leukemia M5 -- Acute Monocytic LeukemiaM5 -- Acute Monocytic Leukemia M6 -- Erythroleukemia (DiGuglielmo’s)M6 -- Erythroleukemia (DiGuglielmo’s) M7 -- Megakaryoblastic LeukemiaM7 -- Megakaryoblastic Leukemia
Myeloperoxidase
(MPO)
p-Phenylene diamine + Catecol + H2O2
MPO > Brown black deposits
Brown deposits considered to be a positive test-differentiates AML from other leukemias
M1 and M2
M3
M5
M4
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 criteriaFAB criteria
MorphologyMorphology CytochemistryCytochemistry
WHO criteriaWHO criteria MorphologyMorphology ImmunophenotypingImmunophenotyping Genetic featuresGenetic features
KaryotypingKaryotyping Molecular testingMolecular testing
Clinical featuresClinical features
WHO Classification of AML
AML with recurrent cytogenic AML with recurrent cytogenic translocationstranslocations
AML with multi-lineage dysplasiaAML with multi-lineage dysplasia AML and myelodysplasia, therapy relatedAML and myelodysplasia, therapy related AML, not otherwise categorizedAML, not otherwise categorized
AML with Recurrent Cytogenetic Translocations (WHO 1995)
t(8;21) -- some maturation of neutrophilic line; t(8;21) -- some maturation of neutrophilic line; rare in older patients; AML1/ETO fusion protein; rare in older patients; AML1/ETO fusion protein; >90% FAB M2>90% FAB M2
t(15;17) -- APL (granular and microgranular t(15;17) -- APL (granular and microgranular variants); retinoic acid receptor (RAR) leukemias; variants); retinoic acid receptor (RAR) leukemias; middle aged adults; DICmiddle aged adults; DIC
inv(16) or t(16;16) -- monocytic and granulocytic; inv(16) or t(16;16) -- monocytic and granulocytic; abnormal eosinophilic componentabnormal eosinophilic component
11q23 -- monocytic; children; most common is 11q23 -- monocytic; children; most common is t(9;11)t(9;11)
Lymphocytic Leukemias
Can involve T or B lymphocytesCan involve T or B lymphocytes B lymphs mature in bone marrowB lymphs mature in bone marrow
Responsible for making antibodiesResponsible for making antibodies T lymphs go to thymus to matureT lymphs go to thymus to mature
Are cytotoxic cells of immune systemAre cytotoxic cells of immune system
FAB Classification of ALL
L1: Small homogeneous blasts; mostly in L1: Small homogeneous blasts; mostly in childrenchildren
L2: Large heterogeneous blasts; mostly in L2: Large heterogeneous blasts; mostly in adultsadults
L3: “Burkitt” large basophilic B-cell blasts L3: “Burkitt” large basophilic B-cell blasts with vacuoleswith vacuoles
L3L2
Periodic Acid Schiff
Periodic acid + Glycogen oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na metabisulfite) > Red deposit
ALL Cytochemistries
Oil Red OOil Red O: stains L3 vacuoles: stains L3 vacuoles Terminal deoxynucleotidyl transferaseTerminal deoxynucleotidyl transferase (Tdt): (Tdt):
DNA polymerase in early lymphoblastsDNA polymerase in early lymphoblasts Cell surface markersCell surface markers (CD’s) (CD’s) Cytoplasmic and surface immunoglobulinsCytoplasmic and surface immunoglobulins: :
B-cell lineB-cell line T-cell receptor T-cell receptor (TCR)(TCR)
WHO Classification of Lymphoproliferative Syndromes
Precursor B Lymphoblastic Leukemia/Lymphoma Precursor B Lymphoblastic Leukemia/Lymphoma (ALL/LBL) -- ALL in children (80-85% of (ALL/LBL) -- ALL in children (80-85% of childhood ALL); LBL in young adults and rare; childhood ALL); LBL in young adults and rare; FAB L1 or L2 blast morphologyFAB L1 or L2 blast morphology
Precursor T ALL/LBL -- 15% of childhood ALL Precursor T ALL/LBL -- 15% of childhood ALL and 25% of adult ALLand 25% of adult ALL
Burkitt Leukemia/Lymphoma (FAB L3)Burkitt Leukemia/Lymphoma (FAB L3)
IndicatorsIndicators FavorableFavorable PoorPoor
WBCWBC < 50,000/< 50,000/LL 50,000/50,000/LL
AgeAge 1 - 101 - 10 < 1 or < 1 or 1010
GenderGender FemaleFemale MaleMale
BlastBlast B-cellB-cell T-cell and mixedT-cell and mixed
KaryotypeKaryotype HyperploidyHyperploidy
Trisomy 4, 10, 17Trisomy 4, 10, 17
t(12;21) (TEL/AML1)t(12;21) (TEL/AML1)
HypoploidyHypoploidy
Trisomy 5Trisomy 5
t(1;19 (E2A/PBX1)t(1;19 (E2A/PBX1)
Mixed lineage leukemiaMixed lineage leukemia
T(9;22) (Ph)T(9;22) (Ph)
BM blast count BM blast count during inductionduring induction
Mkd reduction at day 7Mkd reduction at day 7 Mild reduction at day 7Mild reduction at day 7
Prognosis
Burkitt’s Lymphoma
CML
Typical Labs in CML
Leukocytosis with blastemiaLeukocytosis with blastemia ThrombocytosisThrombocytosis BasophiliaBasophilia Micro-megakaryocytesMicro-megakaryocytes Low LAP score (intermediate if infected)Low LAP score (intermediate if infected) About 10% blasts in BMAbout 10% blasts in BM Philadelphia chromosomePhiladelphia chromosome
Bone marrow aspirateand biopsy
Conventionally, a leukocytosis exceeding Conventionally, a leukocytosis exceeding 50,000 WBC/mm50,000 WBC/mm33 with a significant with a significant increase in early neutrophil precursors is increase in early neutrophil precursors is referred to as a leukemoid reaction.referred to as a leukemoid reaction.
Serum leukocyte alkaline phosphatase is Serum leukocyte alkaline phosphatase is normal or elevated in leukemoid reaction, normal or elevated in leukemoid reaction, but is depressed in chronic myelogenous but is depressed in chronic myelogenous leukemia. leukemia.
Leukemoid reactions are generally benign Leukemoid reactions are generally benign and are not dangerous in and of themselves, and are not dangerous in and of themselves, although they are often a response to a although they are often a response to a significant disease statesignificant disease state
Historically, various clues including the Historically, various clues including the leukocyte alkaline phosphatase score and leukocyte alkaline phosphatase score and the presence of basophilia were used to the presence of basophilia were used to distinguish CML from a leukemoid reaction. distinguish CML from a leukemoid reaction. However, at present the test of choice in However, at present the test of choice in adults to distinguish CML is an assay for the adults to distinguish CML is an assay for the presence of the Philadelphia chromosome, presence of the Philadelphia chromosome, either via cytogenetics and FISH, or via either via cytogenetics and FISH, or via PCR for the BCR/ABL fusion gene. 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 Count 100 consecutive segmented neutrophils and bandsand bands
Score:Score:
0 = no granules0 = no granules
1+ = occasional diffuse granules1+ = occasional diffuse granules
2+ = moderate number of granules2+ = moderate number of granules
3+ = many strongly positive granules3+ = many strongly positive granules
4+ = confluent strongly positive granules4+ = confluent strongly positive granules
0 1+
2+ 3+ 4+
LAP Score
Example:
0 x 35 cells = 01+ x 30 cells = 302+ x 20 cells = 403+ x 10 cells = 304+ x 5 cells = 20
120 LAP Score
Philadelphia Chromosome
9 ;22 translocation almost specific to CML9 ;22 translocation almost specific to CML Karyotype to visualize Ph chromosomeKaryotype to visualize Ph chromosome Produces BCR/c-abl fusion oncogeneProduces BCR/c-abl fusion oncogene Gene product p190 is a hyperactive tyrosine Gene product p190 is a hyperactive tyrosine
kinasekinase Ph chromosome seen in ALL produces p210 and Ph chromosome seen in ALL produces p210 and
chronic neutrophilic leukemia produces p230chronic 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 elderlyExclusive in elderly Lymphocytosis unrelated to viral infectionLymphocytosis unrelated to viral infection Hyper-mature lymphocytes with highly Hyper-mature lymphocytes with highly
condensed nucleicondensed nuclei Smudge cellsSmudge cells
CLLPB and BM
Smudge cell
Bone Marrow
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