1. Leukemia
(FAB/WHO)

2. FM has Leukemia

4. CANCER

5. DEFINE…
Leukemia is defined as:
A. Benign disease of hematopoietic tissue
characterized by replacement of abnormal bone
marrow elements with normal cells
B. Benign disease of hematopoietic tissue
characterized by replacement of normal bone
marrow elements with abnormal cells
C. Malignant disease of hematopoietic tissue
characterized by replacement of abnormal bone
marrow elements with normal cells
D. Malignant disease of hematopoietic tissue
characterized by replacement of normal bone
marrow elements with abnormal cells

10. TRUE or FALSE
 Leukemic cells look different than normal
cells and do not function properly.

15. 4 TYPES of LEUKEMIA
1. Acute Myelogenous (granulocytic)
Leukemia (AML)
2. Chronic Myelogenous (granulocytic)
Leukemia (CML)
3. Acute Lymphocytic (lymphoblastic)
Leukemia (ALL)
4. Chronic Lymphocytic Leukemia (CLL)

16. Acute vs Chronic Leukemia
Acute Chronic
Age Children & young Middle age and
adults elderly
Onset Sudden insidious
Duration weeks to months years
WBC count Variable High

17. Acute vs Chronic Leukemia
Acute Chronic
Platelets Low Early: Normal/ High
Late: Low
Anemia High (>90%) None/ mild
Predomi- Blast cells Mature cells
nant cells
AML = myeloblast CML=granulocytes
ALL= lymphoblast CLL=lymphocytes

18. Acute vs Chronic Leukemia
Acute Chronic
Marrow >20% marrow >70% marrow
cellularity blasts (WHO) cellularity
> 30% marrow (hypercellular);
blasts (FAB) No dysplasia
Diagnosis PBS, BM exam, PBS (peripheral
cytochemical blood smear)
stains, surface
markers,
EM,chromosome

22. TRUE or FALSE
 Blastsare immature blood cells that cannot
carry out the normal function of mature cells.

23. TRUE or FALSE
 Inacute leukemia, blasts are relatively more
mature and they can carry out some normal
functions.

24. TRUE or FALSE
 When leukemia affects myeloid cells, it is
called lymphocytic leukemia.

26. Acute Leukemia

27. TRUE or FALSE
 Acute lymphocytic leukemia (ALL) is the most
common type of leukemia in young children
and it never affects adults.

28. AML vs CML

29. Acute vs Chronic Leukemia

30. Identify….
AML or Normal bone marrow

31. ALL or Normal cells

32. Identify….CLL or Normal cells

33. Identify…. AML or CML

34. Identify… ALL or Normal cells

35. Identify… ALL or CLL

36. Acute Myelogenous Leukemia
(AML)
 acutenonlymphocytic leukemia (ANLL)
 Common form of adult leukemia
 AML blasts do not mature
 Neutropenia, anemia, thrombocytopenia
 Variable WBC count
 Bone marrow blasts >20% (WHO)
 Bone marrow blasts > 30% (FAB)

37. TRUE or FALSE
 Acute myeloid leukemia (AML) is sometimes
called acute nonlymphocytic leukemia
(ANLL).

38. What % of blasts is recommended by the FAB
classification system for a diagnosis of
acute leukemia?
A. 10%
B. 20%
C. 30%
D. 50%

40. AML (+) Bb. Thomson

41. Peripheral Blood Blast Cell

42. Normal bone marrow

43. FAB vs WHO Classification
 French-American-British (FAB) Cx
 Cellular morphology and cytochemical stain
 Acute leukemia as > 30% bone marrow blasts
 Widely used
 World Health Organization Cx
 Cellular morphology and cytochemical stain
 Immunologic probes of cell markers, cytogenetics,
molecular abnormalities & clinical syndrome
 Acute leukemia as > 20% bone marrow blasts
 Standard for diagnosis

44. Acute myeloid leukemias
(AML) Classification - FAB
1. M0: minimally differentiated
2. M1: myeloblastic leukemia without maturation
3. M2: myeloblastic leukemia with maturation
4. M3: hypergranular promyelocytic leukemia
5. M4: myelomonocytic leukemia
6. M4Eo: variant, increase in marrow eosinophils
7. M5: monocytic leukemia
8. M6: erythroleukemia (DiGuglielmo's disease)
9. M7: megakaryoblastic leukemia

45. AML classification - WHO
AML not otherwise categorized
1. AML minimally differentiated
2. AML without maturation
3. AML with maturation
4. Acute myelomonocytic leukemia
5. Acute monocytic leukemia
6. Acute erythroid leukemia
7. Acute megakaryocytic leukemia
8. Acute basophilic leukemia
9. Acute panmyelosis with myelofibrosis

46. STEM CELLS
M0
MYELOID
CELLS
ERYTHROID
CELLS MONOBLAST MYELOBLAST
M6
M6 M5 M1 MEGAKARYOCYTIC
M4 CELLS
M5a M5b
M4E0 M2
M7
PROMYELOCYTE
M3

48. AML

49. Acute Nonlymphocytic Leukemias

50. CYTOCHEMICAL
STAIN
Alpha napthyl
acetate Periodic Acid
Napthol AS-D Schiff (PAS)
Myeloperoxidase Alpha napthyl
Chloroacetate
Sudan Black B butyrate (ALL, M5,
Esterase
(M1-M5) (M4, M5 ) M6,M7)
(M1-M4)
(M6,M7
+ Napthylacetate

51. Chromosomal Leukemia
Abnormality
t(8;21) M2
t(15;17) M3
inv, del, t(16q) M4
t(9;11) M5 (M5a); M4

52. MO: Minimally differentiated
 Undifferentiated Blasts
(No maturation)
 Myeloid phenotype -
CD13, CD33, CD34
 (-) SBB, MPO
 Negative: Auer rods,
Esterase

53. M1 AML without maturation
 > 30% myeloblasts
 Large cells, round nucleus
 Nucleoli (+)
 scanty cytoplasm
 >3% MPO, SBB (+)
 <20% NSE (+)
 CD 13, 33, 117

54. M1 AML without maturation

55. Which FAB classification shows 3% (+) MPO,
SBB, shows <20% (+) with nonspecific
esterase and contains primarily myelobalst
with distinct nucleoli?
A. M1
B. M2
C. M3
D. M4

56. M2 AML with maturation
 Common type
 >30% myeloblasts
 >10% granulocyte
 Kidney shape nucleus
 Nucleoli (+)
 (+) Auer rods
 Eosinophilic granules
 >50% MPO, SBB (+)
 CD 13, 33

57. M2 AML with maturation

58. Auer Rods

59. Promyelocyte constitute 10% of acute leukemia
with >50% of leukemic cells positive for
peroxidase and Sudan black B. What is the
FAB classification?
A. M1
B. M2
C. M3
D. M4

60. M3 (hypergranular promyelocytic)
 Promyelocyte-predominant
 Large, kidney shape
 (+) Auer rods (faggot cells)
 basophilic, bilobed nuclei
 CD 13,33
 High incidence of DIC

61. Acute myeloid leukemia with very
abnormal cells (AML M3/ t15;17)

62. M4 Acute myelomonocytic
 >30% myeloblast (FAB)
 >20% granulocyte
 >20% promonocytes and
monocytes
 CD 11, 13, 33,14
 (+) Auer rods common
 High serum lysozyme level
 M4Eo = w/ eosinophilia

63. The serum lysozyme level was greatly
increased in this patient. Cells are positive
for CD11 and CD14 by flow cytometry.
Which disease is most likely?
A. Acute myeloblastic leukemia
B. Promyelocytic leukemia
C. Myelomonocytic leukemia
D. Undifferentiated leukemia

64. M5: acute monocytic leukemia
1. M5a – without maturation
 Monoblasts , few promonocytes
1. M5b – with maturation
 Blast, Promonocytes (BM), Monocytes (Blood)

66. M5a
 Monoblast ameboid with
round to oval nuclei,
 prominent nucleoli,
 <20% promonocytes/mono
 Vacuolated cytoplasm

67. AML M5a

68. M5b
 > 20% promonocytes,
monocytes
 Promonocytes folded,
convulated nucleus
 Azurophilic granules

69. AML M5b

70. M6 - erythroleukemia
 Large, bizarre,
round-to-oval cells
 (+) nucleoli
 > 50% Erythroblasts
 > 30 % Myeloblasts
 CD 45,71 Glycophorin A
 CD 13, 15,33 myeloblast
 PAS (+)

71. M6 (erythroblast)

72. M6 (erythroblast)

73. M7 – acute megakaryoblastic
 >30% megakaryoblasts
 platelet like granules on
PAS stain
 NSE (but not BE) (+)
 Myeloid blasts may
show SBB or MPO (+)
 CD 41,42,61

74. M7 (Megakaryoblast)

75. Megakaryoblast

76. Chronic
Myeloproliferative
Diseases (MPD)

77. Classification of chronic
myeloproliferative diseases (MPD)
FAB WHO
1. Chronic myelogenous CML Ph+: t(9;22)(qq34;q11),
leukemia (CML) BCR/ABL
Chronic neutrophilic leukemia
2. Polycythemia vera (PV) Polycythemia vera (PV)
3. Essential thrombocytemia (ET) Essential thrombocytemia (ET)
4. Chronic Myelofibrosis Chronic idiopathic myelofibrosis

78. Myeloproliferative disease (MPD)
Disorder Cell Type Feature
CML Granulocytes (Ph1), LAP/NAP, Splenomegaly
PV Erythrocytes RBC, Splenomeg, Teardrop,
High LAP
ET Platelets Thrombocytosis
CMF Fibroblast NRBC, LAP, Dacrocytes

79. Chronic Myelocytic Leukemia
 < 5% blasts
 M:E ratio 10:1
 Mature granulocyte
 all stages of maturation (+)
 High WBC,Splenomegaly
 Enlarged platelets
 95% (+) Philadelphia (Ph)
chromosome t(9;22)
 Low LAP score
 High LAP (leukemoid)

80. Chronic Myeloid Leukemia

81. Leukemoid CML
reaction
WBC High High
Anemia (-) (+)
PBS Shift to the Left Shift to the left (blast)
Toxic granulation Eosinophilia,
Dohle bodies basophilia
LAP score High Low
Philadelphia (-) (+)
chromosome

82. Chromosomal Leukemia
Abnormality
t(9;22) - Philadelphia CML
chromosome

83. What is the M:E ratio in patients with chronic
myelogenous leukemia?
A. 1:5
B. 1:10
C. 10:1
D. 3:1

84. What is the most common chromosomal
abnormality found in chronic myelogenous
leukemia?
A. t(8;14)
B. t(9:22)
C. t(1;12)
D. Trisomy 12

85. Polycythemia Vera
 Malignant hyperplasia of myeloid stem cell
 Increase erythrocytes
 High blood viscosity (BP)
 Secondary polycythemia- High RBC (high EPO)
 Normal: Plasma volume, WBC, Platelet ormal
 Relative (pseudo) polycythemia
 High Hgb
 Normal EPO,RBC mass, WBC, platelet

86. Essential Thrombocythemia
 Proliferationmegakaryocytes (adults)
 High platelet count, giant form
 Platelet function abnormalities
 Leukocytosis
 Usually without hepatomegaly and
splenomegaly

87. Chronic myelofibrosis
 Myeloid stem cell disorder
 Proliferation of erythroid, granuclocytic,
megakaryotic precursor in marrow
 Marrow fibrosis
 Immature neutrophils, nucleated RBC
 5% blasts, anemia, marked poikilocytosis,
nucleated RBC
 Bleeding, hepatosplenomegaly

88. A peripheral blood stained smear showed 5%
blasts, anemia, poikilocytosis and nucleated
RBC. What condition should be suspected?
A. Chronic myelogenous leukemia
B. Acute myelogenous leukemia
C. Thalassemia major
D. myelofibrosis

89. Myelodysplastic
Syndromes(MDS)

90. Myelodysplastic Syndromes
 Clonal proliferation of hematopoietic cells,
including erythroid, myeloid, and
megakaryocytes
 Blood cytopenia (anemia)
 30% blasts in bone marrow (FAB)
 Monocytosis, ringed sideroblast, macrocytosis

91. All of the following are characteristic of
myelodysplastic syndrome EXCEPT:
A. Lymphocytosis
B. Monocytosis
C. Ringed sideroblast
D. macrocytosis

92. Myelodysplastic syndromes
(MDS)- FAB Classification
1. Refractory Anemia (less than 5% blasts)
2. Refractory Anemia with Ringed Sideroblasts
(RARS) (Sideroblastic anemia)
3. Refractory Anemia with Excess Blasts
(RAEB) (5 to 20% blasts)
4. Refractory Anemia with Excess Blasts in
Transformation (RAEB-T) (20 to 30% blasts)
5. Chronic Myelomonocytic Leukemia (CMML)
(more than 1000 monocytes/mm3)

93. Myelodysplastic
syndromes (MDS)
(FAB)
< 5% myeloblast > 5% myeloblast
RAEB-T
RA RARS RAEB
( 21-30%
(6-20%
myeloblast
myeloblast
chronic myelomonocytic leukemia monocytosis
(CMML) = 20% myeloblast splenomegaly

96. RARS
>15% ringed sideroblast (PB)

97. RAEB-T (vacuolated blast)
 >20% marrow blasts
 <30% peripheral blast
 WHO- acute leukemia
instead MDS
 FAB - CMML

98. RAEB-T
vacuolation granular & clot like

99. Refractory Anemia
 Not responsive to therapy
 Oval macrocytes, reticulocytopenia
 bone marrow blast <5%
 peripheral blast <1%

100. Chronic Myelomonocytic
Leukemia (CMML)
 5-20% bone marrow blast
 <5% peripheral blood blast
 Absolute monocytosis
 Leukocytosis

101. Refractory Anemia with excess
blasts (RAEB)
 5-20% bone marrow blast
 <5% peripheral blood blast
 Common cytopenias
 Absolute monocytosis

102. Acute lymphocytic
leukemia (children)

104. Acute Lymphocytic Leukemia
French-American-British (FAB)
Classification
L1 ALL, child
L2 ALL, adult
L3 ALL, (Burkitt)

105. WHO classification- Cytogenetics
Leukemia Chromosomal
Abnormality
ALL-pre B t(1;19)
ALL-T t(11;14)
ALL-Burkitt’s lymphoma t(8;14), t(2;8), t(8;22)

106. Immunophenotyping
Types FAB TdT Tc Ag Bc Ag
Precursor B L1,L2 +, - - +
Precursor T L1,L2 +, + + -
B-cell L3 -,- - +

108. Acute Lymphocytic Leukemia
FAB Class L1
 small , uniform cells
 scanty cytoplasm
 absent nucleoli
 70% c-ALL
20% T-ALL
rarely B-ALL or null-ALL
 TdT (+)
 PAS (+)

109. Acute Lymphocytic Leukemia
FAB Class L2
 Large, heterogenous
 basophilic cytoplasm
 Nucleoli present
 50% c-ALL
30% T-ALL
 TdT (+)
 PAS (+)

110. Acute Lymphocytic Leukemia
FAB Class L3 (Burkitt’s)
 large varied cells
 deeply basophilic
 vacuolated cytoplasm
 Nucleoli (+)
 Usually B-ALL
 TdT (-)
 PAS (+)

111. Which of the following stain/reaction show a
positive result in most patient with acute
lymphocytic leukemia?
A. Sudan black and peroxidase
B. Chloroacetate esterase
C. Non specific esterase
D. Terminal deoxynucleotidyl transferase

112. The leukemia commonly associated with
pediatric age group is:
A. Acute myeloblastic leukemia
B. Acute lymphoblastic leukemia
C. Chronic lymphoblastic leukemia
D. Chronic myelocytic leukemia

113. Chronic Lymphocytic
Leukemia (CLL)
B cell malignancy (CD19,20)
 Male adult
 Autoimmune hemolytic anemia
 Lymphocytosis, homogenous, small,
hyperclumped lymphocytes and smudge cells
 neutropenia, thrombocytopenia, Ig
 Small lymphocytic lymphoma
 Lymphadenopathy, splenomegaly,
hepatomegaly

114. Chronic Lymphocytic
Leukemia (CLL)

115. Chronic Lymphocytic
Leukemia (CLL)

116. Hairy Cell Leukemia (HCL)
 B cell malignancy (CD
19, CD20)
 Marked splenomegaly
 Pancytopenia
 Cytoplasm show hair
like projection
 (+) TRAP

117. Hairy Cell Leukemia (HCL)

118. Hairy Cell Leukemia (HCL)

119. Hairy Cell Leukemia (HCL)

120. Prolymphocytic Leukemia
B cell or T cell malignancy
 Marked splenomegaly
 Lymphocytosis, prolymphocytes
 Anemia, thrombocytpenia

121. Plasma Cell Dyscrasia

122. Multiple Myeloma
 Plasma (lymphoid)/ B lymphocyte neoplasm
 >30% plasma cells (BM)
 Skeletal system tumor of plasma cell (myeloma)
 Monoclonal gammopathy disease
 Excess IgG / IgA production
 electrophoresis: M spike in gamma globulin
 Bence Jones portein (free light); kidney damage
 High viscosity; rouleaux

123. Multiple myeloma

124. Normal BM / MM in BM

125. Plasma Cell Leukemia
 Abnormal plasma cells in blood
 Pancytopenia
 Rouleaux
 Monoclonal gammopathy

126. Waldenstrom’s
macroglobulinemia
 malignancy involving excess B-lymphocytes
that secrete immunoglobulins
 Excessive IgM (viscous)
 No bone tumor
 Lyphadenopathy, hepatosplenomegaly
 M spike in gamma globulin region

127. Lymphomas

128. Lymphomas
 Malignant cells in solid lymphatic tissue
 Localized to BM to blood
 Lyphadenopathy
 CD, PCR, Tissue biopsy
 Hodgkin
 B cell
 T/NK cell (non Hodgkin)
 Sezary syndrome

129. Hodgkin Lymphoma
 EBV associated
 (+) Reed Strenberg
cells = large
multinucleated cells
with large nucleoli
 Mild anemia,
eosinophilia,
monocytosis
 High LAP, ESR

130. DIAGNOSTIC
PROCEDURES

133. Leukemia Diagnosis
1. Blood Test – CBC
2. Differential blood count – immature “blast”
3. Hematocrit assay- proportion of RBC blood
4. Hgb assay- O2 carrying pigment
5. Blood coagulation- clotting
6. Blood morphology and staining – cell shape,
structure, nucleus (PBS)

134. Leukemia Diagnosis
7. Blood chemistry
 ALP = CML diagnosis
 Vitamin B12= CML
 Ca, K, phosphate, uric acid = ALL

136. 1. Enzyme cytochemical staining
5 blood smear/bone marrow smear
Air dried
Papenheim/May Grunwald Giemsa (MGG)/
MPO / NSE

137. Cytomorphology
Pappenheims
MPO
NSE

138. Cytochemical stain
1. Myeloperoxidase (MPO) stain
 (+) AML (granulocyte, monocyte, Auer rods)
 (-) ALL
1. Sudan Black B ( phospholipids, proteins)
 (+) AML (granulocyte, monocyte, Auer rods)
 (-) ALL

139. Cytochemical stain
3. Esterases
 Specific esterase stain (napthol AS-D
chloroacetate esterase stain) = granulocyte
 (+) FAB M1, M2, M3, M4
 (-) FAB M5
 Non specific esterase stain (alpha napthyl
acetate and alpha napthyl butyrate) = monocyte
 (+) FAB M5
 (-) FAB M1, M2, M3, M4

140. Cytochemical stain
4. Periodic Acid Schiff (PAS)
 (+) ALL
 (+) FAB M6 (erythrolekemia)
5. Leukocyte Alkaline Phosphatase (LAP)
 Low LAP score = CML
 High LAP score = NLR
 Normal LAP score= Hodgkin

141. Cytochemical stain
6. Tartrate Resistant acid Phosphatase (TRAP)
 (+) hairy cell leukemia
7. Perl’s Prussian Blue stain
(+) Siderocytes with iron inclusions
(siderocytic granules/pappenheimer bodies)
(+) HA, beta thallasemia major, sideroblastic Anemia
(+) Sideroblasts = nRBC with iron granules
Ringed sideroblasts= iron encircle nucleus
(+) MDS (Refractory anemia, RARS, sideroblastic
anemia)

142. A positive non specific esterase stain indicates
differentiation into which cell type?
A. Monocytic
B. Lymphoid
C. Megakaryocytic
D. Plasmacytoid

143. Which cytochemical stain is used to detect
acute myelogenous leukemia M7?
A. Terminal deoxynucleotidyl transferase (TdT)
B. Myeloperoxidase (MPO)
C. platelet peroxidase
D. Sudan black b (SBB)

144. 2. Immunophenotyping
 Principle: Highly specific Ags on cell
surfaces are detected by monoclonal Abs
tagged with fluorescein and the complex are
determined by flow cellcytometry
 Examination of the proteins on cell surfaces
and the antibodies

145. Immunophenotyping
Types FAB TdT Tc Ag Bc Ag
c Ag s Ag
Precursor L1,L2 +, - - + -/+ -
B
Precursor T L1,L2 +, + + - - -
B-cell L3 -,- - + + -/+
TdT = Terminal deoxynucleotidyl transferase
CAg= common Ag
Sag= surface antigen

146. CD markers
B cell= C19, CD20
 T cell = CD2, CD3, CD5
 Myeloid= CD13, CD14, CD33

147. 3. Multiparameter flow cytometry
EDTA heparinized BM or blood
Cell lysis/ Ficoll hypaque gradiation
MFC

148. 4. CYTOGENETICS
 test to look for certain changes of the
chromosomes (genetic material) of the
lymphocytes
 Chromosomal analysis
 t(9;22) = Ph1 CML
 t(15;17) = M3

149. CYTOGENETICS
1. Culture malignant hematopoietic tissue to
obtain dividing cells in the metaphase stage
where the mitotic process is stopped
2. Cells are fixed
3. Cells are dropped into microscope slide
where chromosome fall in a random pattern
4. Stained with giemsa or quinicrine
5. Chromosome banding (G band or Q band)

150. Cytogenetics
 Metaphase after G-
banding in a c-ALL with
t(9;22) so called
Philadelphia
translocation

151. 5. Fluorescence in situ
hybridization
cyto smears of bone marrow or peripheral blood
metaphases and interphase nuclei
fixation 2 h time for hybridization
APL detecting PML-RARA fusion signals in interphase-FISH
interphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24-
color FISH or comparative genomic hybridization (CGH)
Hybridize overnight
interphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24-
color FISH or comparative genomic hybridization (CGH)

152. Metaphase before and after analysis by 24-color FISH in a case
with AML and complex aberrant karyotype

153. 5. Molecular Mtd- PCR
Ficoll Hypaque density gradient centrifugation
for DNA or RNA preparation
sequencing or even gene expression profiling

154. end na po…