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TRANSLOCATIONS
o Most common type of DNA change that can lead to leukemia
o A part of one chromosome breaks off and becomes attached to a different chromosome
o The point at which the break occurs affect nearby genes which can turn on oncogenes or turn off
genes that help a cell to mature
o Examples
t(1;22) Acute Megakaryoblastic Leukemia (FAB M7)
t(8;21) Acute Myeloblastic Leukemia with Maturation (FAB M2)
t(9;11) Acute Monocytic Leukemia (FAB M5)
t(15;17) Acute Promyelocytic Leukemia (FAB M3)
t(8;22) Burkitt-Type Lymphocytic Leukemia (FAB L3)
t(2;8) FAB L3
t(8;14) Burkitt’s Lymphoma and T-Cell Leukemia/Lymphoma
t(11;14) Multiple Myeloma and Chronic Lymphocytic Leukemia
t(10;14) T-Cell Leukemia/Lymphoma
DELETIONS
o May result in the cell losing a gene that helped keep its growth in check
o Examples
6q deletion – ALL and and Non-Hodgkin’s Lymphoma
INVERSIONS
o A part of a chromose gets turned around causing a reversed order which can result in the loss of
genes because of failure to read instructions in protein translation
o Examples
inv(16) – Acute Myelomonocytic Leukemoa with Eosinophilia (FAB M4Eo)
inv(3) – in Myelodysplatic Syndrome andl Acute Myeloid Leukemias except M3
(Promyelocytic)
ADDITION
o Extra chromosome or part of a chromosome is gained
o Lead to many copies of a gene within a cell (problematic if it the are oncogenes)
ONCOGENES
o Single oncogene in a cell is not sufficient to convert the cell into a full-blown cancer cell
o Examples
Abl – Chronic Myelogenous Leukemia
Abelson Murine Leukemia viral oncogene
Myc – Burkitt’s Lymphoma
Overexpressed due to t(8;14)
A transcription factor and controls overexpression of several genes
MYC familiy of oncogene may be activated by gene rearrangement such as the
t(8;14) or amplification
Ras – Leukemias
A normal gene that can be converted into an oncogene via point mutation in
most cases
When ras transcription is increased an excess of the gene’s protein is in the cell,
and the positive signals for cell division begin to outweigh the negative signals
BCL-2
B-Cell Lymphoma Gene 2
BCL2 prevents apoptosis hence overexpression leads to overexpression of cells
PROTOONCOGENES
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
o Antecedents of oncogenes
o Central regulators of growth in normal cells
TUMOR SUPRESSING GENES
o Anti-oncogenes
CELLULAR FACTORS that induce end-stage differentiation
o B-Interferon
o Tumor Growth Factor
o Tumor Necrosis Factor
HAZARDOUS AGENTS
OCCUPATIONAL
o Ionizing Radiations – increased incedence of leukemia
o Acute and Chronic forms of Myelogenous Leukemia are most frequently associated with radiation
o Chemicals used in laboratory
o Chemists – risk of cancer involving lymphatic system
o Radiologists – cancer involving bone marrow
o Rubber-industry worker – cancer involving the blood
o Woodworkers – cancer involving the lymphatic system
ENVIRONMENTAL
o Non-Ionizing Radiations
CHEMICAL/DRUG
o Benzene – Leukemias and Aplastic Anemia
o Increased risk of AML (higher risk than ALL)
o Platinum-based treatment for ovarian cancer – risk of secondary leukemia
VIRAL AGENTS
o Epstein-Barr Virus (EBV)
o Human T-Cell Leukemia Virus 1 (HTLV-I)
ACUTE LEUKEMIAS
Cytoplasm
CYTOPLASM
FAB NAME NUCLEUS Nucleolus CHROMATIN
Abundance
Color and NOTES
Granule
AML not otherwise
Myeloblastic (min. Fine to Coarse
M0 None catgorized
differentiated)
SCANT
Common in <18 mos
Myeloblastic w/o Round to oval Nucleus
M1 variable M:F 1:1
maturation Single to multiple distinct
Nucleolus Surv : 3.5 mos
azurophillic Middle aged
Myeloblastic granules 40% in > 60 y/o
M2 MODERATE
with/without
With maturation M>F 1.6 : 1
Auer rods Surv 8.5 mos
Promyelocytic
Round to Single to Azurophillic Most aggressive
indented to multiple Granules
Bleeding tendency
M3 Myelocytic lobed cottage- (granules FINE PROMINENT with
loaf may multiple Median age (38)
obscure) auer rods M>F 2:1
Surv 16 mos
Risk of leukostasis
Pharyngitis
Gingival hyperplasia
blue to gray
>50 y/o
M4 Myelomonoblastic MODERATE maybe
granulated Most predom secodary
hema malignancy in MM
Round to indented fold Nucleus M > F 1.4 : 1
Single to Multiple distinct
Nucleolus Risk of leukostasis
M5a – Monoblastic
gray-blue
Variable lacy In young adults (16)
SCANT TO dustlike
M5 Monoblastic or ropy
MODERATE lavender
M<F 0.7 : 1
granules M5b – Monocytic
In mid age (49)
M>F 1.8 : 1
Erythremic myelosis
Single to bizarre
Open DiGuglielmo’s
multinucleated
M6 Erythroblastic multilobed Single to
megaloblastoid ABUNDANT red to blue > 50 y/o
Multiple M>F 1.4 : 1
Distinct Surv 11 months
Single to gray blue Organomegaly in children
SCANT TO
M7 Megakaryoblastic Round to Oval Moderately
MODERATE
with Cytopenia
Vacuolated blobbing CD41 and CD61; -antiMPO
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
SUDAN BLACK B
o Differentiation of Acute Myelogenous Leukemia and Acute Myelomonocytic Leukemia from Acute
Lymphocytic Leukemias
o Binds irreversibly to an undefined granule component in granulocytes eosinophils and monocytes
o Reaction product black granular
o Reagents
Fixative : 40% Formaldehyde solution’s vapor
SBB in absolute ethanol
Phenol Buffer
May Grunwald Giemsa or Lieshman Stain as Counterstain
o Postive Reactions : Granulocytes – sudanophillic as they mature
Myeloblasts
Few small granules in golgi region
Promyelocyte – increased granulation
Neutrophilic Myelocytes
Granules concentrated near nucleus or at the rim of cytoplasm
Metamyelocytes / bands/ segmenters
Strongly Positive
Eosinophils
Positve at periphery of granules (seen in all stages of its development)
Clear core of eosinophil granules
Monocytic cells
Granules scattered over entire cell
o Basophils exhibit variable reaction (bright red or purple staining)
o Negative Reactions : Lymphocytic Lineage – sudanophobic
Also erythrocytic lineage and Megakaryocytic-thrombocytic lineage
MPO negative Neutrophils
o Leukemias + in SBB Cytochemical Reaction (1, 2, 4 , 5)
M1 and M2
M2 strongly positive
Variable
M4 and M5
o Rare cases of ALL show non-granular smudge positivity not seen with MPO
MYELOPEROXIDASE STAIN
o for differentiating AML blasts from ALL blasts
o marker for primary granules and auer rods
o PERFORM ONLY ON FRESH SPECIMEN
o Reaction product is brown and granular
o Reagents
Fixative : Buffered formol acetone
Hydrogen peroxide
Hematoxylin as countersaatin
DAB substrate
Buffer 7.3 (Sorenson’s Phosphate buffer)
o Positive Reactions : Granulocytes (Promyelocytes and Metamyelocytes strongly reacting)
Neutrophils except blasts
Strongly positve
Monocytes except blasts
Faint reaction with few granules
Eosinophils
Strongly positive granules with large eosinophilic granule that is easily
distinguished from netrophil granules
o Variable : megakaryocytic-thrombocytic cells
o Negative
Erythrocytic cells
Plasma cells
Lymphocytic cells
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
Basophils
Blasts
o Leukemias + in MPO Cytochemical Reaction (1, 2, 4, 5 and 7)
M1 and M2
M2 ++
M4 and M5 variable
M7 + in unfixed samples
PERIODIC ACID-SCHIFF
o Also for differentiating AML from ALL
o Useful for supporting diagnosis of DiGuglielmo’s (M6) as it stains erythrocytes of M6
Erythrocytic lineage does not react on PAS except for erythorcytes of M6
o Oxidizes 1-2 glycol groups to produce a stable dialdehyde which give a red product when
exposed to leucobasic fuchsin (Schiff’s reagent)
o Reagents
Fixative : Methanol
Periodic Acid 1%
Schiff’s Reagent
Aqueous Hematoxylin as counterstain
o Reaction product is diffused-pink or large-red aggregates
o Postive
Neutrophils except blast forms
Megakaryocytes and its blasts in Malignant or Proliferative Disease
Erythrocytes of Fab M6
o Variable Ractions
Eosinophils, Basophils
Granules are negative but cytoplasm may contain faintly PAS + granules
Monocytes
Faint pink cytoplasm
May have granules
Lymphocytes
May contain few positive granules
Thrombocytes
Intense pink or red
Megakaryocytes
Diffuse pink or red with coarse red pigments
Leukemic Lymphoblasts of L1 OR L2
Coarse or block-like positivity
o Negative Reactions (blasts may show diffuse reaction ocassionally)
Erythrocytic Cells
Myeloblasts
Monoblasts
o Leukemias + in PAS Cytochemical Reaction
M5, M6, M7
M5 ++
L1 or L2 +/-
NAPTHOL AS-D CHLOROACETATE ESTERASE (LEDER STAIN)
o Use for identifying cells of the granulocytic series
o Specific Esterase stain
o Reaction Product is bright red
o Confined to cells of neutrophil series and mast cells
o Myeloblasts stain rarely while promyelocytes show strong positivity
o Reagents
Fixative : Buffered Formol Acetate
Buffer pH 7.4
Napthol AS-D Chloroacetate Substrate solution in N,N-Dimethyl-formamide
Coupling reagent : Hexazotized new fuchsin and Sodium nitrate solution
Counterstain : Hematoxylin
o Positive Reaction
Promyelocyte
Myelocyte
Metamyelocyte
Bands and Segmenters
o Leukemias + with NASDCA Cytochemical Reaction
M2, M6, M7 = +
M4 is +/-
ALPHA-NAPTHYL ESTERASE
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
o Non-specific esterase
o Marker for monocytes, megakaryocytes, and plasma cells
o Reagents
Fixative : Buffered Formol Acetone
Phosphate Buffer pH 8.0
A-napthyl butyrate in acetone
Coupling reagent : Fast Garnet GBC
Counterstain : Hematoxylin
o Reaction product is brown and granualr
o Postive
Monocytes stain strongly
Stain negative when incubated with sodium fluoride
Histiocytes
Erythroblasts
Megakaryoblasts
o Leukemias + with aNA Cytochemical reaction
M4, M5, M6, M7
M4 and M5 ++
M6 and M7 +++
Acid-Phosphatase Stain
o Acid Phosphatase is present in all hematopoietic cells and are found in lysosomes
o Stains lymphocytic cells = for ALL
o For diagnosis of T-Cell Leukemia
o Tartrate Resistance
Indicative of Hairy Cell Leukemia
o Leukemias
ALL esp L1 and L2
Negative for AMLs
Leukocyte Alkaline Phosphatase
o Distinguishes CML from leukemoid reactions and myeloproliferative disorders
o Reagents
4% Formalin Methanol as fixative
Substrate is Napthol AS Phosphate
Tris Buffer pH 9
Fast blue bb salt for coupling
Neutral red as counter stain
o Reaction product is blue and granular
o LAP score
Determined by evaluation of staining intensity (0 to 4+) of 100 neutrophils/bands
0 = negative, no granules
1+ = ocassional granules scattered in cytoplasm
2+ = moderate
3+ = numerous
4+ = heavy positivity with numerous coarse granules frequently overlying nucleus
Normal Lap score ranges from 15 to 130
< 15 Lap Score
CML
PNH
MDS
> 130 Lap Score
Infections
Growth Factor therapy
Myeloproliferative disorders other than CML
Inflammatory disorders
Pregnancy
Oral contraceptives
Stess
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
IMMUNOLOGICAL MARKERS
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
B-CELL MALIGANCIES
o Monoclonal B-Cell Lymphocytosis (MLD)
Preced virtually all cases of CLL/SLL
High Count MLD resembles Rai stage 0
o B-Cell Prolmphocytic Leukemia
Rare lymphoid neoplasms with poor prognosis
Requires more than 55% of circulating lymphoid cells to have a morphology of
prolymphocyte
Occurs in older adults (age 69)
B-PLL lymphocyte morphology
Round nucleus with moderate cytoplasm and a distinct punched-out nucleolus
CD+
CD19
CD20
CD22
CD4
t(8,14) and del 17p
no t(11;14) – differentiates it from mantle cell lymphoma
CMYC and TP53 mutation
o Hairy Cell Leukemia
Uncommon
Mature B-cell malignancy
BRAF V600E – genetic lesion
Affects 30 y/o and above
M>F
Morphology
Fine hair-like irregular cytoplasmic projections
TRAP +
Markers
CD19
CD20
CD22
CD24
CD25
Interlukin-2 (Tac)
Strong Sig
o Hairy Cell Leukemia variant (vHCL)
More aggressive
vHCL cells are smaller with a central round nucleus, prominent nucleoli, larger N:C ratio
and basophilic cytoplasm
poor prognosis
o Non-Hodgkin Lymphomas
lord.wynlor | 9
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
HODGKIN DISEASE
o Reed-Sternberg Cells – arise from a single clone, a common B-cell precursor
Aneuploidy – characteristic feature
Gain of chromosomes
1, 2, 5, 12, and 21
Structural rearrangements of chrom 1 more frequently observed
T-CELL and NK CELL MALIGNANCIES
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
MYELOPROLIFERATIVE NEOPLASMS
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
References
Graeter, L. J., In Hertenstein, E. G., In Accurso, C. E., & In Labiner, G. H. (2015). Elsevier's medical laboratory
science examination review.
McKenzie, S. B., Williams, J. L., & Landis-Piwowar, K. (2015). Clinical laboratory hematology.
McPherson, R. A., Pincus, M. R., & Henry, J. B. (2017). Henry's Clinical diagnosis and management by laboratory
methods. St. Louis: Elsevier.
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