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CLINICAL
HEMATOLOGY
(WBC)
LECTURE BY:
JAMES PATRICK C. PICAR, RMT, MSMLS(c)
TRIKS PICAR, RMT

LEUKOPOIESIS:Production of Leukocytes
•Leukopoiesis is stimulated by interleukins and colony-
stimulating factors (CSFs)
-Interleukins are numbered (e.g., IL-1, IL-2),
whereas CSFs are named for the WBCs they stimulate
(e.g., granulocyte-CSF stimulates granulocytes)
•Macrophages and T cells are the most important
sources of cytokines
•Many hematopoietic hormones are used clinically to
stimulate bone marrow
TRIKS PICAR, RMT

TRIKS PICAR, RMT
MYELOID SERIES
LYMPHOID SERIES

FORMATION OF LEUKOCYTES
•All leukocytes originate from hemocytoblasts
•HemocytoblastsàMyeloid Stem Cells & Lymphoid Stem Cells
•Myeloid stem cells àMyeloblast/Monoblast
•Lymphoid stem cells àLymphoblasts
•MyeloblastsàEosinophils, Neutrophils, or Basophils
•Monoblastsàmonocytes
•Lymphoblastsàlymphocytes
TRIKS PICAR, RMT

HEAMOCYTOBLAST
MYELOID
STEM CELL
MYELOBL
AST
PROMYELO
CYTE
MYELOC
YTE
METAMYEL
OCYTE
BAND
FORM
GRANULO
CYTE
GRANULOPO
IESIS
TRIKS PICAR, RMT

Large spherical nucleus, azurophilicor primary cytoplasmic
granules(only produced by these cells), no subtypes ( can't
recognize cell lines at this stage)
Spherical nucleus, heterochromatic, specific or secondary
granules (seen only in granulocytes) begin to form, can
divide
Easily recognizable lineages, kidney bean –shaped nucleus
Not seen in Eosinophilor Basophillineage, also called stab
form, horse-shoe shape nucleus, up to 3% in peripheral
blood
Mature forms
Earliest recognizable cell in marrow, large euchromatic
nucleus, nucleoli, basophilic cytoplasm
GRANULOPOEISIS -
TRIKS PICAR, RMT

MYELOID LINEAGE
1.MYELOBLAST
% IN THE BM: 0-3%
SIZE: 14-20um
NUCLEUS:
SHAPE: ROUND
N/C RATIO: 8:1-4:1
COLOR: REDDISH PURPLE
CHROMATIN: DELICATE AND DISPERSE
NUCLEOLI: 2-3
TRIKS PICAR, RMT

MYELOID LINEAGE
1.MYELOBLAST
CYTOPLASM:
COLOR: PALE TO DEEP BLUE
CONTENTS: varying amounts
of granules
CLINICAL CONDITIONS:
•M0,M1,M2,M3,M4,M6
•MYELODYSPLASTIC SYNDROMES:
RAEB, RAEB-t
•MYELOPROLIFERATIVE SYNDROMES:
CML, MMM
TRIKS PICAR, RMT

MYELOID LINEAGE
1.MYELOBLAST
üTYPE I MYELOBLAST
-high N/C ratio of 8:1-4:1
-slightly basophilic cytoplasm,
fine nuclear chromatin
-NUCLEOLI: 2-4 VISIBLE
NUCLEOLI
-has no visible granules when
observed with ROMANOWSKY
STAIN
TRIKS PICAR, RMT

MYELOID LINEAGE
1.MYELOBLAST
üTYPE II MYELOBLAST
-presence of dispersed PRIMARY/
AZUROPHILIC GRANULES in the
cytoplasm
üTYPE III MYELOBLAST
-darker chromatin and more
purple cytoplasm
-contains >20 granules but do
not obscure the nucleus
-rare in normal bone marrow
TRIKS PICAR, RMT

MYELOID LINEAGE
2.PROMYELOCYTE
% IN THE BM: 1-5%
SIZE: 16-25um (18-25um)
NUCLEUS:
SHAPE: ROUND OR OVAL, ECCENTRIC
N/C RATIO: 2:1
COLOR: PURPLE
CHROMATIN: relatively fine becoming coarser
NUCLEOLI: 2-3
TRIKS PICAR, RMT

MYELOID LINEAGE
2.PROMYELOCYTE
CYTOPLASM:
COLOR: EVENLY BASOPHILIC/
BLUE
CONTENTS: FULL OF PRIMARY
OR AZUROPHILIC
GRANULES
CLINICAL CONDITIONS:
üM2, M3
üMYELOPROLIFERATIVE SYNDROMES:
CML, MMM
üGROWTH FACTOR THERAPY
üSEVERE INFECTIONS
TRIKS PICAR, RMT

TRIKS PICAR, RMT

MYELOID LINEAGE
3.NEUTROPHILIC MYELOCYTE
% IN THE BM: 6-17%
SIZE: 12-18um
NUCLEUS:
SHAPE: ROUND, OVAL, OR FLATTENED ON ONE SIDE
N/C RATIO: 3:1-3:2
COLOR: DARK PURPLE
CHROMATIN: coarser chromatin pattern
NUCLEOLI: none visible
TRIKS PICAR, RMT

MYELOID LINEAGE
3.NEUTROPHILIC MYELOCYTE
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
variable number of non-specific granules
CLINICAL CONDITIONS:
üM2
üMYELOPROLIFERATIVE SYNDROMES: CML, MMM
üGROWTH FACTOR THERAPY
üSTRESS
TRIKS PICAR, RMT

MYELOID LINEAGE
3.NEUTROPHILIC MYELOCYTE
üEARLY MYELOCYTES
-very similar to promyelocyte in size and nuclear characteristics
-patches of grainy pale pink cytoplasm representing secondary granules begin to be evident in the area of Golgi apparatus (DAWN OF NEUTROPHILIA)
üLATE MYELOCYTES
-smaller than promyelocyte
-nucleus has considerably more heterochromatinTRIKS PICAR, RMT

TRIKS PICAR, RMT

MYELOID LINEAGE
4.NEUTROPHILIC METAMYELOCYTE
% IN THE BM: 3-20%
SIZE: 14-16um (10-15 um)
NUCLEUS:
SHAPE: TYPICAL KIDNEY-BEAN, PEANUT-SHAPED OR SLIGHTLY INDENTED
N/C RATIO: 7:3-1:1
COLOR: DARK PURPLE
CHROMATIN: coarse, blue-black chromatin
NUCLEOLI: ABSENT
TRIKS PICAR, RMT

MYELOID LINEAGE
4.NEUTROPHILIC
METAMYELOCYTE
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
pinkish to reddish-blue
granules
NOTE:
Synthesis of tertiary granules or
gelatinase granules may begin
TRIKS PICAR, RMT

MYELOID LINEAGE
5.NEUTROPHILIC
BAND CELL
% IN THE BM: 9-32%
% IN THE PERIPHERAL BLOOD: 0-5%
SIZE: 9-12um
NUCLEUS:
SHAPE: BAND-SHAPED/ SAUSAGE-SHAPED/ MARKEDLY INDENTED/ HIGHLY CLUMPED; indentation is greater than ½ of the width of round nucleus
N/C RATIO: 1:1-1:2
COLOR: PINKISH-BLUE
CHROMATIN: coarse, blue-black chromatin
TRIKS PICAR, RMT

MYELOID LINEAGE
5.NEUTROPHILIC BAND CELL
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
pinkish to blue granules
NOTE:
•During this stage, actual segmentation has not yet occurred
•INCREASED BAND CELL COUNT: DIAGNOSTIC OF PX WITH INFECTIONTRIKS PICAR, RMT

TRIKS PICAR, RMT

MYELOID LINEAGE
6.SEGMENTED NEUTROPHILS/ POLYMORPHONUCLEAR NEUTROPHILS(PMN’s)
% IN THE BM: 7-30%
% IN THE PERIPHERAL BLOOD: 50-70%
SIZE: 9-12um
NUCLEUS:
SHAPE: 2-5 lobes connected by a threadlike filament; nuclear indentation is >1/2 of its diameter
N/C RATIO: 1:3-1:5
COLOR: DARK PURPLE
CHROMATIN: heavily clumped
NUCLEOLI: NONE
TRIKS PICAR, RMT

MYELOID LINEAGE
6.SEGMENTED
NEUTROPHILS/
POLYMORPHONUCLEAR
NEUTROPHILS(PMN’s)
CYTOPLASM:
COLOR: LIGHT PINK TO BLUISH
CONTENTS: many small evenly
distributed pink to
rose-violet granules
CLINICAL CONDITION:
*INFECTIONS
TRIKS PICAR, RMT

TRIKS PICAR, RMT

NEUTROPHIL KINETICS
•NEUTROPHIL PRODUCTION: 0.9-1.0 X 10 ꝰcells/kg/day
•TRANSIT TIME FROM MYELOBLAST THROUGH MYELOCYTE: 6 DAYS
•TRANSIT TIME THROUGH THE MATURATION POOL: 4-6 DAYS
•HALF-LIFE: approximately 7 HOURS
•G-CSF: stimulates granulocyte release from the bone marrow
•MAC-1: trigger the death and phagocytosis of neutrophils
•INTEGRINSand SELECTINS: are of significant importance in allowing
neutrophils to marginate as well as exit the blood and enter the
tissues by a process known as DIAPEDESIS
TRIKS PICAR, RMT

NEUTROPHIL FUNCTION
MAJOR FUNCTION:
PHAGOCYTOSIS and destruction of foreign material and microorganisms
SECONDARY FUNCTION:
GENERATION OF NEUTROPHIL EXTRACELLULAR TRAPS (NETs)
*NETs: extracellular threadlike structures
: able to trap and kill G+ and G-bacteria as well as fungi
: generated at the time that neutrophils die as a result of antibacterial activity
*NETosis: unique form of neutrophil cell death that results in the release of NETs
TERTIARY FUNCTION: SECRETORY FUNCTION
: source of Transcobalamin I or R binder protein
TRIKS PICAR, RMT

Leukocytes
Distribution-blood and CT (as transient or wandering cells)
Function-immune protection, movement (cell motility)
Diapedesis-movement out of blood into connective tissue
Chemotaxis-movement directed by homing molecules
TRIKS PICAR, RMT

MYELOID LINEAGE
3.EOSINOPHILIC MYELOCYTE
SIZE: 12-18um
NUCLEUS:
SHAPE: ROUND, OVAL, OR FLATTENED ON ONE SIDE
N/C RATIO: 3:1-3:2
COLOR: DARK PURPLE
CHROMATIN: coarser chromatin pattern
NUCLEOLI: none visible
TRIKS PICAR, RMT

MYELOID LINEAGE
3.EOSINOPHILIC
MYELOCYTE
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
Numerous large, round
specific granules staining
orange-brown to orange-
blue; variable numbers of
nonspecific granules
CLINICAL CONDITIONS:
üEOSINOPHILIC LEUKEMIATRIKS PICAR, RMT

TRIKS PICAR, RMT
LARGE GRANULES WITH
CRYSTALLINE CORE

MYELOID LINEAGE
4.EOSINOPHILIC METAMYELOCYTE
% IN THE BM: 3-20%
SIZE: 14-16um (10-15 um)
NUCLEUS:
SHAPE: TYPICAL KIDNEY-BEAN, PEANUT-SHAPED OR SLIGHTLY INDENTED
N/C RATIO: 7:3-1:1
COLOR: DARK PURPLE
CHROMATIN: coarse, blue-black chromatin
NUCLEOLI: ABSENT
TRIKS PICAR, RMT

MYELOID LINEAGE
5.EOSINOPHILIC
BAND CELL
% IN THE BM: 9-32%
% IN THE PERIPHERAL BLOOD: 0-5%
SIZE: 9-12um
NUCLEUS:
SHAPE: BAND-SHAPED/ SAUSAGE-SHAPED/ MARKEDLY INDENTED/ HIGHLY CLUMPED; indentation is greater than ½ of the width of round nucleus
N/C RATIO: 1:1-1:2
COLOR: PINKISH-BLUE
CHROMATIN: coarse, blue-black chromatin
TRIKS PICAR, RMT

MYELOID LINEAGE
5.EOSINOPHILIC
BAND CELL
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
numerous orange-red granules
TRIKS PICAR, RMT

TRIKS PICAR, RMT

MYELOID LINEAGE
6.EOSINOPHILS
% IN THE BM: 1-3%
% IN THE PERIPHERAL
BLOOD: 1-3%
SIZE: 9-12um
NUCLEUS:
SHAPE: 2 lobes usually
N/C RATIO: 1:3-1:5
COLOR: DARK PURPLE
CHROMATIN: heavily clumped
NUCLEOLI: NONE TRIKS PICAR, RMT

MYELOID LINEAGE
6.EOSINOPHILS
CYTOPLASM:
COLOR: PINK-BLUE
CONTENTS: many large, round,
uniform reddish-orange
granules
CLINICAL CONDITIONS:
üPROTOZOAN INFECTIONS
üHYPEREOSINOPHILIC SYNDROME
üALLERGIC DISORDERS
üCML
üDERMATITIS
üHODGKIN’S LYMPHOMA
TRIKS PICAR, RMT

MYELOID LINEAGE
3.BASOPHILIC
MYELOCYTE
SIZE: 12-18um
NUCLEUS:
SHAPE: ROUND, OVAL, OR
FLATTENED ON ONE SIDE
N/C RATIO: 3:1-3:2
COLOR: DARK PURPLE
CHROMATIN: coarser
chromatin pattern
NUCLEOLI: none visibleTRIKS PICAR, RMT

MYELOID LINEAGE
3.BASOPHILIC
MYELOCYTE
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
few large specific granules
staining dark bluish-purple
to bluish-black
CLINICAL CONDITIONS:
üBASOPHILIC LEUKEMIA
TRIKS PICAR, RMT

TRIKS PICAR, RMT

MYELOID LINEAGE
4.BASOPHILIC METAMYELOCYTE
SIZE: 14-16um (10-15 um)
NUCLEUS:
SHAPE: TYPICAL KIDNEY-BEAN, PEANUT-SHAPED OR SLIGHTLY INDENTED
N/C RATIO: 7:3-1:1
COLOR: DARK PURPLE
CHROMATIN: coarse, blue-black chromatin
NUCLEOLI: ABSENT
TRIKS PICAR, RMT

MYELOID LINEAGE
4.BASOPHILIC
METAMYELOCYTE
CYTOPLASM:
COLOR: PINKISH BLUE
CONTENTS:
Few large, dark-blue black
granules
TRIKS PICAR, RMT

MYELOID LINEAGE
5.BASOPHILIC
BAND CELL
SIZE: 9-12um
NUCLEUS:
SHAPE: BAND-SHAPED/ SAUSAGE-SHAPED/ MARKEDLY INDENTED/ HIGHLY CLUMPED; indentation is greater than ½ of the width of round nucleus
N/C RATIO: 1:1-1:2
COLOR: DARK PURPLE
CHROMATIN: coarse, blue-black chromatin
NUCLEOLI: NONE
TRIKS PICAR, RMT

MYELOID LINEAGE
5.BASOPHILIC
BAND CELL
CYTOPLASM:
COLOR: PINK BLUE
CONTENTS:
few large dark bluish-purple to
bluish-black granules
TRIKS PICAR, RMT

MYELOID LINEAGE
6.BASOPHILS
% IN THE BM: <1%
% IN THE PERIPHERAL
BLOOD: 0-2%
SIZE: 9-12um
NUCLEUS:
SHAPE: 2 lobes usually
obscured by granules
N/C RATIO: 1:3-1:5
COLOR: DARK PURPLE
CHROMATIN: heavily clumped
NUCLEOLI: NONE TRIKS PICAR, RMT

MYELOID LINEAGE
6.BASOPHILS
CYTOPLASM:
COLOR: PINK-BLUE
CONTENTS: numerous, dark
blue-black granules
CLINICAL CONDITIONS:
üBASOPHILIC LEUKEMIA
üMYELOPROLIFERATIVE SYNDROMES
üALLERGY AND INFLAMMATION
üINFECTION
TRIKS PICAR, RMT

MYELOID LINEAGE
7.MAST CELLS
SIZE: 9-12um
NUCLEUS:
SHAPE: ROUND
N/C RATIO: 1:1
COLOR: DARK PURPLE
CHROMATIN: heavily clumped
NUCLEOLI: NONE
TRIKS PICAR, RMT

MYELOID LINEAGE
7.MAST CELLS
CYTOPLASM:
COLOR: DARK-BLUE
CONTENTS: many dark blue
granules
CLINICAL CONDITIONS:
üMAST CELL DISEASE
TRIKS PICAR, RMT

HEAMOCYTOBLAST
MYELOID STEM
CELL
MYELOMONOB
LAST
PROMONOC
YTE
MONOCYT
E (BLOOD)
MACROPHA
GE
(TISSUES)
MONOPOEI
SIS
MONOCYTE-
MACROPHAGE
SERIES
TRIKS PICAR, RMT

MONOCYTIC SERIES
1.MONOBLAST
SIZE: 12-20um
NUCLEUS:
SHAPE: ROUND/OVAL
N/C RATIO: 6:1
COLOR: RED PURPLE
CHROMATIN: fine, well
distributed
NUCLEOLI: 1-2
TRIKS PICAR, RMT

MONOCYTIC SERIES
1.MONOBLAST
CYTOPLASM:
COLOR: BLUE
CONTENTS: clear
CLINICAL CONDITION:
üM4a and M4b
üM5
TRIKS PICAR, RMT

MONOCYTIC SERIES
2.PROMONOCYTE
SIZE: 12-20um
NUCLEUS:
SHAPE: OVAL/INDENTED
N/C RATIO: 5:1
COLOR: LIGHT PURPLE
CHROMATIN: fine, well
distributed
NUCLEOLI: 1-2
TRIKS PICAR, RMT

MONOCYTIC SERIES
2.PROMONOCYTE
CYTOPLASM:
COLOR: BLUE
CONTENTS: clear
CLINICAL CONDITION:
üM4a and M4b
üM5
TRIKS PICAR, RMT

MONOCYTIC SERIES
2.MONOCYTE
SIZE: 14-20um
NUCLEUS:
SHAPE: DEEPLY INDENTED,
KIDNEY-BEAN SHAPE
N/C RATIO: 3:1
COLOR: LIGHT PURPLE
CHROMATIN: loose, coarse linear
pattern
NUCLEOLI: ABSENT
TRIKS PICAR, RMT

MONOCYTIC SERIES
3.MONOCYTE
CYTOPLASM:
COLOR: DULL, GRAY BLUE
CONTENTS: fine, non-
specific ground glass
appearance with vacuoles
TRIKS PICAR, RMT

TRIKS PICAR, RMT

HEAMOCYTOBLAST
LYMPHOID STEM
CELL
LYMPHOBLAST
PROLYMPHOCYTE
LYMPHOCYTE
PLASMA CELLS
LYMPHOPOI
ESIS
LYMPHOID
SERIES
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
1.LYMPHOBLAST
SIZE: 10-22um
NUCLEUS:
SHAPE: ROUND OR OVAL,
CENTRAL OR ECCENTRICAL
N/C RATIO: 7:1-4:1
COLOR: REDDISH-PURPLE
CHROMATIN: fine, lacy pattern to
moderately coarse
NUCLEOLI: 1-2 (prominent)
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
1.LYMPHOBLAST
CYTOPLASM:
COLOR: MODERATE TO
DARK BLUE
CONTENTS: smooth, no granules,
occasional vacuoles
CLINICAL CONDITIONS:
üL1, L2,L3
üLYMPHOBLASTIC LEUKEMIA
üBURKITT’S LYMPHOMA
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
2.PROLYMPHOCYTE
SIZE: 10-18um
NUCLEUS:
SHAPE: OVAL, SLIGHTLY
INDENTED
N/C RATIO: 5:1
COLOR: DARK PURPLE
CHROMATIN: coarse and
clumped
NUCLEOLI: 1-2
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
2.PROLYMPHOCYTE
CYTOPLASM:
COLOR: LIGHT BLUE TO
DARK BLUE
CONTENTS: few azurophilic
granules
CLINICAL CONDITIONS:
üM4a and M4b
üM5
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
3.LYMPHOCYTE
SIZE: 7-15um
NUCLEUS:
SHAPE: ROUND OR SLIGHTLY
INDENTED, ECCENTRIC
N/C RATIO: 3:1
COLOR: DEEP PURPLE-BLUE
CHROMATIN: coarse and
clumped
NUCLEOLI: none visible
TRIKS PICAR, RMT

LYMPHOCYTIC SERIES
3.LYMPHOCYTE
CYTOPLASM:
COLOR: SKY BLUE TO
DEEP BLUE
CONTENTS: scant and usually
non-granular; few
azurophilic granules may
be seen
TRIKS PICAR, RMT

PLASMACYTIC SERIES
1.PLASMABLAST
SIZE: 12-14um
NUCLEUS:
SHAPE: ROUND
N/C RATIO: 5:1-4:1
COLOR: PURPLISH-RED
CHROMATIN: fine and has linear
strands
NUCLEOLI: one or more
TRIKS PICAR, RMT

PLASMACYTIC SERIES
1.PLASMABLAST
CYTOPLASM:
COLOR: BLUE
CONTENTS: non-granular
CLINICAL CONDITION:
üPLASMA CELL LEUKEMIA
TRIKS PICAR, RMT

PLASMACYTIC SERIES
2.PROPLASMACYTE
SIZE: 12-15um
NUCLEUS:
SHAPE: ROUND,
ECCENTRICALLY PLACED
N/C RATIO: 5:1-4:1
COLOR: PURPLISH-RED
CHROMATIN: moderately
clumped
NUCLEOLI: 0-2
TRIKS PICAR, RMT

PLASMACYTIC SERIES
2.PROPLASMACYTE
CYTOPLASM:
COLOR: DARK BLUE, prominent light areas next to nucleus
CONTENTS: non-granular
CLINICAL CONDITION:
üPLASMA CELL LEUKEMIA
üMULTIPLE MYELOMA
üWALDENSTROM’S MACROGLOBULINEMIA
üRESPONSE TO INFECTION
TRIKS PICAR, RMT

PLASMACYTIC SERIES
3.PLASMA CELL
SIZE: 9-20um
NUCLEUS:
SHAPE: OVOID AND
ECCENTRICALLY PLACED ,
WITH A “WHEELSPOKE
PATTERN”
COLOR: DARK PURPLE
NUCLEOLI: NONE
TRIKS PICAR, RMT

PLASMACYTIC SERIES
3.PLASMA CELL
CYTOPLASM:
COLOR: DEEP BLUE
CONTENTS: with occasional
bluish granules
CLINICAL CONDITION:
üPLASMA CELL
DYSCRASIAS
üRESPONSE TO INFECTION
TRIKS PICAR, RMT

MONONUCLEAR CELLS
•Includes:
üMONOCYTES
üLYMPHOCYTES
*BOTH have nuclei that are not segmented but
are round, oval, indented, or pooled
NOTE:
*NORMAL WBC DIFFERENTIAL IN ADULTS:
4.5 –11.5 x 10ꝰ/L
*KINETICS
-movement of cells through developmental
stages, into the circulation, and from the
circulation to the tissues
TRIKS PICAR, RMT

NON-MALIGNANT
LEUKOCYTE DISORDERS
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NUCLEAR ABNORMALITIES
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QUALITATIVE DISORDERS OF LEUKOCYTES
Pelger-Huet Anomaly (PHA)
•Also known as: TRUE PHA/ CONGENITAL
PHA
•Inheritance pattern: Autosomal
Dominant
•Characteristic: DECREASED nuclear segmentation (bilobed/unilobed); with
coarse chromatin pattern affecting all
WBCs but most obvious in mature
neutrophil
•Mutation in: Lamin B-receptor Gene
•Appearance of nucleus:
round, ovoid/dumbbell , peanut shaped
•MOST COMMON GENETIC
DISORDER OF WBC’S
•Neutrophil function: NORMAL
Note:
Lamin b-receptor
§Plays a major role in leukocyte
nuclear shape changes that
occurs during normal maturation
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
Pelger-Huet Anomaly (PHA)
2 TYPES
HETEROZYGOUS
•Appearance of nucleus
(neutrophils): round, ovoid,
peanut shaped
•Bilobed forms: Spectacle-like/
Pince-nez
•55-93% of neutrophils are affected
HOMOZYGOUS
•Appearance of nucleus:
round
•ALL neutrophils are
affected and demonstrate
ROUND nuclei
Note:
Neutrophils in Pelger-Huet
Anomaly appear to function
NORMALLY
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
Pseudo Pelger-Huet Anomaly (PHA)
•Also known as: Acquired PHA
•May developed in cases of
MYELODYSPLASTIC
DISORDERS: AML, CML,
PRELEUKEMIA, Patients with
infections or tumors that
metastasized to the bones,
HIV infection, tuberculosis, M.
pneumoniaeand severe
bacterial infectionsand drugs
•Appearance of nucleus:
Round
•Cytoplasm: Hypogranular
Note:
Drugs:
ümycophenolate mofetil
üvalproate,
üsulfisoxazole,
üganciclovir,
üibuprofen,
üchemotherapies (paclitaxel and docetaxel)
TRIKS PICAR, RMT

PELGER-HUET
ANOMALY
TRIKS PICAR, RMT

HOW TO DISTINGUISH
TRUE/CONGENITAL AND PSEUSO/ACQUIRED
PELGER-HUET ANOMALY
TRUE/CONGENITAL
PELGER-HUETANOMALY
PSEUDO/ACQUIRED
PELGER-HUET ANOMALY
NUMBER OF CELLS
PRESENT WITH PHA
MORPHOLOGY
63%-93%<38%
WBCLINEAGES
AFFECTED
ALL WBC’sNEUTROPHILS ONLY
GRANULATIONOF
NEUTROPHILS
NORMALGRANULATIONHYPOGRANULAR
Summarized from: Rodak’s, 5thEd. page 476TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
Neutrophil Hypersegmentation
•Normally: 3-5 lobes
•Neutrophils have more than
5 lobes
•Inheritance pattern:
Autosomal dominant
•Most often associated with
Megaloblastic Anemia
Hereditary Hypersegmentation
•Inheritance pattern: Atusomaldominant
•Patients are Asymptomatic
•Assoc. condition: myelokathexis, Undritzanomaly
Acquired Hypersegmentation
•Associated with megaloblastic anemia, folate deficiency and pernicious anemia
Note:
Myelokathexis
§Rare hereditary condition characterized by normal granulocyte production; however there is impaired release into the circulation leading to neutropenia
§Neutrophils appear hypermature
§Mutation: CXCR4 receptor
TRIKS PICAR, RMT

NEUTROPHIL
HYPERSEGMENTATION
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CYTOPLASMIC
ABNORMALITIES
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QUALITATIVE DISORDERS OF LEUKOCYTES
Alder-Reilly Anomaly
•Inheritance pattern: Autosomal Recessive
•Characterized by: presence of abnormally large, darkly staining azurophilic granules (Alder-Reilly bodies/Reilly bodies) composed primarily of digested mucopolysaccharides resembling toxic granulation
•Granules maybe seen in: lymphocyte and monocyte
•BASIC DEFECT: incomplete degradation of mucopolysaccharides
•WBC function: NORMAL
•Associated with: Hunter,
Hurler, Sanfilippo, and
Maroteaux-Lamy
polydystrophicdwarfism
TRIKS PICAR, RMT

ALDER-REILLY ANOMALY
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
Chediak-Higashi Syndrome
•Inheritance pattern: Autosomal Recessive
•Characterized by abnormal fusion of granules in most of the granulated cells throughout the body
•Hematologic findings: giant lysosomal granules in granulocytes, monocytes and lymphocytes
•Fused granules result in leukocyte dysfunction and recurrent pyogenic infections
•Platelet lack DENSE
granules
•Associated with: ALBINISM
•Accelerated stage:
hepaatomegaly, liver
failure, lymphadenopathy
with lymphoma-like
morphology, neuropathy
and death
•Mutation: CHS1 LYST gene
TRIKS PICAR, RMT

CHEDIAK-HIGASHI SYNDROME
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
May-Hegglin Anomaly
•Inheritance pattern: Autosomal dominant
•Characteristics: variable thrombocytopenia, giant platelets, large Dohle-like inclusions in neutrophils, eosinophils, monocytes, and basophils
•Mutation in:MYH9 gene
•Majority of individual are ASYMPTOMATIC; sometimes with mild bleeding tendencies
•Dohle bodies: pale blue, spindle-shaped inclusions from rough endoplasmic reticulumTRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
Chronic Granulomatous Disease (CGD)
•Rare, inherited disorder caused
by decreased ability of
phagocytes to produce
superoxide and reactive oxygen
species
•Characterized by defects in the
RESPIRATORY BURST OXIDASE
SYSTEM
•Cells engulf but are UNABLE TO
KILL microorganisms
•Mutation: gp91phox or p47 gene
CLIN. FINDINGS:
üRecurrent chronic bacterial
and fungal infections of the
LUNGS, SKIN, LYMPH NODES,
and LIVER
üFormation of granuloma
(can destruct hollow organs)
üRECURRENT PNEUMONIA:
most common cause of
death
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
Chronic Granulomatous Disease (CGD)
2 WAYS OF EVALUATION
1.CHEMILUMINESCENCE/
FLOW CYTOMETRY
•Uses DIHYDRORHODAMINE-123
2. NITROBLUE TETRAZOLIUM TEST (NBT TEST)
•Evaluates respiratory burst by reduction of the nitroblue tetrazolium dye inside phagocytes
NOTE:
NITROBLUE TETRAZOLIUM TEST
•PRINCIPLE: reduction of a yellow,
water-soluble nitroblue tetrazolium
dye
•REAGENT: Nitroblue Tetrazolium Dye
•COLOR OF REAGENT: YELLOW
•(+): BLUE
•(-): YELLOW
TRIKS PICAR, RMT

NITROBLUE TETRAZOLIUM BLUE DYE TEST
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
Leukocyte Adhesion Disorders (LAD)
•INHERITANCE PATTERN:
Autosomal Recessive
•Inability of neutrophils and
monocytes to adhere to
endothelial cells and to
migrate from blood to tissues
TYPES
LAD I
•Mutation in genes responsible
for B2 integrin subunits
Note:
B2 integrins: needed for neutrophil
adhesion to endothelial cells,
recognition of bacteria, and outside-in
signaling
•CLIN. FINDINGS:
üRecurrent infections
üNeutrophilia
üLymphadenopathy
üSplenomegaly
üSkin lesions
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
Leukocyte Adhesion Disorders (LAD)
LAD II
•Rarer than LAD I
•Leukocytes have NORMAL B2 integrin
•MUTATION: SLC35C1 gene
CLIN. FINDINGS:
üRecurrent infections
üNeutrophilia
üPhysical growth retardation
üCoarse face and/or other physical deformities
üNeurological defects
LAD III
•Very rare, AUTOSOMAL
RECESSIVE
•Mutation: eeeeeeeeKINDLIN-3
(needed for leukocyte rolling)
CLIN. FINDINGS:
üMild LAD I-like immunodeficiency
üRecurrent bacterial and fungal
infections
üIncreased risk of bleeding
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
JOB’S SYNDROME
•Also known as: HyperimmunoglobulinemiaE
•RANDOM MOVEMENT of phagocytes is NORMAL; but DIRECTIONAL MOTILITY/ CHEMOACTIVITY is IMPAIRED
•Cells respond slowly to chemotactic agents
CLIN. FINDINGS:
üPersistent boils
üRecurrent “cold” staphylococcal abscesses
üMarkedly increased: _______
üSkeletal abnormalities
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
LAZY LEUKOCYTE SYNDROME
•BOTH RANDOM AND DIRECTED
MOVEMENT OF THE CELLS ARE
DEFECTIVE
•Bone marrow reserves of
granulocytes are NORMAL, but
release to the peripheral
blood is POOR
•Cells contain defective ACTIN
FILAMENTS
CLIN FINDINGS:
üNeutropenia (constant finding)
üLow-grade fever
üRecurrent infections involving the
gums, mouth, and ears
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
CONGENITAL C3 DEFICIENCY
•INHERITANCE PATTERN:
autosomal recessive
autosomal recessivee
•MOST SEVOMPLEMENT
DEFICIENCY
CLIN. FINDINGS:
üSevere recurrent infections with
encapsulated bacteria
üGLOMERULONEPHRITIS
Note:
C3
•Most abundant complement
component
•Most important complement in
the activation of the
complement pathways
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(NORMAL MORPHOLOGY WITH FUNCTIONAL ABNORMALITIES)
MYELOPEROXIDASE (MPO) DEFICIENCY
•INHERITAN •Mutation: _____________
•BACTERIAL KILLING is SLOWED but COMPLETE
*ACQUIRED MYELOPEROXIDASE DEFICIENCY
üHematologic neoplasms
üLead Poisoning
Note:
Myeloperoxidase
•Stimulates production of oxidant agents that attack phagocytized microbes
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
A. MUCOPOLYSACCHARIDE/ GLYCOSAMINOGLYCAN STORAGE DISEASES
•Also known as: MUCOPOLYSACCHARIDOSES
•Family of inherited disorders of GAG degradation
•INHERITANCE PATTERN: Autosomal recessive
•Cells in affected tissues are swollen and have apparent ballooning and cleaning of cytoplasm
•Partially degraded GAG
builds up in the lysosomes
and results in:
üPhysical abnormality
üMental retardation
•(+) ALDER-REILLY BODIES/
REILLY BODIES
•(+) PURPLE: using TOLUIDINE
BLUE
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
TYPES OF MUCOPOLYSACCHARIDE/ GLYCOSAMINOGLYCAN STORAGE DISEASES
TYPENAMEDEFICIENT ENZYME
MPSI-SEVEREHURLER SYNDROMEa-l-iduronidase
MPS I-ATTENUATED/
MPS V
SCHEIE SYNDROMEa-l-iduronidase
MPS II-SEVEREHUNTER SYNDROMEIduronatesulfatase
MPS IIISanfilippo Syndrome Type A
SanfilippoSyndrome Type B
Sanfilippo Syndrome Type C
HeparanN-sulfatase
a-N-acetylglucosaminidase
Acetyl CoA:a-glucosaminideN-
acetyltransferase
MPSIVMorquioSyndrome Type A
MorquioSyndrome Type B
Galactose-6-sulfatase
b-galactosidase
MPS VIMaroteaux-LamySyndrome
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
B. LIPID STORAGE DISEASES
•Also known as: LIPIDOSES
•Macrophages of one or more
tissues become overloaded with
lipids
•LIPID CATABOLISM IS DEFECTIVE
•Inheritance pattern: Autosomal
recessive
1.GAUCHER’S DISEASE
•MOST COMMON of the
LIPIDOSES
•Cause: lack of b-glucosidase
•Common in: Ashkenazi Jews
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
CLINICAL SUBTYPES OF GAUCHER’S
DISEASE
TYPE I GAUCHER’S DISEASE
•Also known as: Non-neuropathic adult
•Clinical onset: Childhood/Adulthood
•HEPATOSPLENOMEGALY: (+)
•SKELETAL ABNORMALITIES: (+)
•NEURODEGENERATION: (-)
•DEATH: VARIABLE
•ETHNIC PREDILECTION: common in
ASHKENAZI JEWS
TYPE II GAUCHER’S DISEASE
•Also known as: Acute
Neuropathic/Infantile/Cerebral
•Clinical onset: Infancy
•HEPATOSPLENOMEGALY: (+)
•SKELETAL ABNORMALITIES: (-)
•NEURODEGENERATION: (+++)
•DEATH: by 2 YEARS
•ETHNIC PREDICLECTION: PAN-
ETHNIC
•MORE SEVERE FORM OF
GAUCHER’S DISEASE
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
TYPE III GAUCHER’S DISEASE
•Also known as: Subacute
neuropathic/Juvenile
•Clinical onset: Childhood
•HEPATOSPLENOMEGALY: (+)
•SKELETAL ABNORMALITIES: (+)
•NEURODEGENERATION: (++)
•DEATH: during 2ndto 4thdecade of
life
•ETHNIC PREDILECTION: SWEDES
CLINICAL FINDINGS (IN GENERAL)
üHepatosplenomegaly
üIncreased serum phosphatases
ü(+) GAUCHER CELLS IN BM
üNORMO/NORMO ANEMIA
üLEUKOPENIA
üTHROMBOCYTOPENIA
Note:
GAUCHER CELLS
•Found in the bone marrow
•Large macrophage with small, eccentric nucleus with striated/ wrinkled appearance described as: crumpled tissue paper/onion-like skin/ chicken-scratch appearance
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
Note:
Chitotriosidase: useful test in determining the level of glucocerebrosidein storage
PSEUDO-GAUCHER CELLS
•Result of excessive cell turn-over and overwhelming of glucocerebrosidaseenzyme
•Found in BM of patients with:
üThalassemia
üCML
üAML
TREATMENT
üENZYME REPLACEMENT
THERAPY WITH RECOMBINANT
GLUCOCEREBROSIDASE
üSTEM CELL TRANSPLANT
TRIKS PICAR, RMT

GAUCHER CELLS
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
2. NIEMANN-PICK DISEASE
•Inheritance pattern: Autosomal
recessive
•Abnormal accumulation of
SPHINGOMYELIN and CHOLESTEROL
in mononuclear phagocytic cells
and some parenchymal cells
•DEFICIENCY: SPHINGOMYELINASE
•More commonly seen in ASHKENAZI
JEWS
CLIN. FINDINGS:
üHEPATOSPLENOMEGALY
üPOOR PHYSICAL
DEVELOPMENT
ü(+) FOAM CELLS/ PICK CELLS
Note:
FOAM CELLS/ PICK CELLS
•Abnormal macrophage whose
cytoplasm is filled with many
small lipid droplets
•Increased numbers may lead to
MACROPHAGE OVERLOAD
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
NIEMANN-PICK DISEASE SUBTYPES
TYPES A AND B
•MUTATION: SMPD 1 GENE
•DEFICIENCY: ACID
SPHINGOMYELINASE
•Subsequent build-up of
sphingomyelin in the LIVER,
KIDNEYS, and LUNGS (BRAIN also
affected in TYPE A)
•(+) NIEMANN-PICK CELL IN BM
•Disease is often FATAL by 3 YEARS
OF AGE
TYPE A: <5% normal
sphingomyelinase activity
TYPE B: 10%-20% normal
sphingomyelinase activity
TYPE C
•Decreased in cholesterol
effluxing from the intracellular
endosome/ lysosome of cytosol
•With SYTEMIC, NEUROLOGIC
and PSYCHIATRIC SYMPTOMS
•Most patients die before 25
years old
TRIKS PICAR, RMT

NIEMANN-PICK DISEASE
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
(MONOCYTE/MACROPHAGE LYSOSOMAL STORAGE DISEASES)
3. TAY-SACHS DISEASE
•Accumulation of glycolipid and
gangliosides
•(+) VACUOLATED CYTOPLASM
•Deficiency: HexoaminidaseA
4. SANDHOFF’S DISEASE
•Deficiency: HexoaminisaseA
and B
5. SEA-BLUE HISTIOCYTOSIS
•INHERITANCE PATTERN:
Autosomal recessive
•Syndrome marked by:
üHepatosplenomegaly
üThrombocytopenia
üAccumulation in the spleen
and bone marrow of histiocytes
filled with lipid-rich granules
than stain BLUE-GREEN (using
Wright’s or Giemsa stain)
TRIKS PICAR, RMT

SEA-BLUE HISTIOCYTES
TRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
GENETIC B AND T LYMPHOCYTE ABNORMALITIES
•Genetic disorders that generally result in the decreased production of B cells, T cells or both.
T LYMPHOCYTE ABNORMALITY
1.DIGEORGE SYNDROME
•Absence or underdevelopment of thymus
•Markedly decreased number of T lymphocytes
•Microdeletion of: Chromosome Band 22q11:2
•Complete DiGeorgeSyndrome: athymicindividuals
B LYMPHOCYTE ABNORMALITY
1.SEX-LINKED AGAMMAGLOBULINEMIA (XLA)
•B cell disease most frequently caused by mutation in the gene encoding for Bruton Tyrosine Kinase (BTK): without BTK, B-lymphocytes fail to reach maturity and will die prematurelyTRIKS PICAR, RMT

QUALITATIVE DISORDERS OF LEUKOCYTES
COMBINED B LYMPHOCYTE/T
LYMPHOCYTE ABNORMALITIES
1. SEVERE COMBINED
IMMUNODEFICIENCY (SCID)
•More common type of
combined B and T lymphocyte
abnormality
•Mutation: IL-2 receptor gamma
chain
•Results in depletion of T cells, B
cells and NK cells
2. WISKOTT-ALDRICH SYNDROME
•Mutation: WASp
•CLIN. FINDINGS:
üLow levels or absence of
Wiskott-Aldrich protein
üImmunodeficiency
üEczema
üThrombocytopenia
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
NEUTROPHILS
•Neutrophilia is suspected when
the neutrophil count of the
patient is above 7.0 x10ꝰ/L
(ADULTS) and above 8.5 x10ꝰ/L
•Normal relative neutrophil count is
between: between 50-70%
•Normal Absolute Neutrophil
Count: 1.7 to 7.5 x 10^9/L
•Increased levels: Neutrophilia
•Decreased levels: Neutropenia
Note:
Leukemoid Reaction
•Reactive leukocytosis above 50 x 10_, with neutrophilia and a marked shift to the left
•Mostly a result of: acute and chronic infection, metabolic disease, inflammation, or response to malignancy
•May be confused with CHRONIC MYELOGENOUS LEUKEMIA (CML)
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
HOW TO DISTINGUISH CML FROM
LEUKEMOID REACTION
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
Note:
Leukoerythroblastic reaction
•Presence of neutrophils,
nucleated red blood cells, and
teardrop cells in the same
sample
•Possible presence of space-
occupying lesions in the bone
marrow
•Strongly associated with
PRIMARY MYELOFIBROSIS
Note:
Neutropenia
•Decrease in the absolute neutrophil count below 2.0
x10ꝰ/L (white adults)and
below1.3 x10ꝰ/L (black adults)
•The risk of infection increases as the ANC lowers, especially below 1.0 x10ꝰ/L
Agranulocytosis
•Refers to neutrophil count of
less than 0.5x10ꝰ/L
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
Classifications of Neutropenia
1.Alloimmune Neonatal Neutropenia
•Maternal IgG crosses the
placenta and binds to neutrophil-
specific antigens inherited from
the father
2. Autoimmune Neutropenia in Children
•Moderate to severe neutropenia
developing as a result of
antibodies to HNA-1
•Self-limiting and resolves after 7 to
24 months
3. Secondary Autoimmune Neutropenia
•Associated with autoimmune disorders: rheumatoid arthritis, Felty syndrome, SLE and Sjogren’s syndrome
4. Congenital Severe Neutropenia
•Consist of Kostmannsyndrome (infantile agranulocytosis): severe neutropenia that presents shortly after birth and bone marrow granulocyte hypoplasia
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
Classifications of Neutropenia
5. Chronic Idiopathic Neutropenia
•Predominantly affects
women 18 to 35 years of
age
•Bone marrow produces
more immature neutrophils
•Treatment: G-CSF
6. FanconiAnemia
7. DyskeratosisCongenita
8. Shwachmann-Bodian-Diamond
Syndrome
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
EOSINOPHILS
•Major function: DEGRANULATION
•Eosinophilia is suspected when the
absolute eosinophil count of the patient is above 0.4 x 10ꝰ/L
•Normal relative count: 1%-3%
•Normal AEC: 0-0.3% x 10ꝰ/L
2 BROAD CATEGORIES (EOSINOPHILIA)
•Underdeveloped areas: parasite
infestation (helminthes and
protozoa)
•Developed countries: allergic
reactions
Note:
Hypereosinophilic Syndrome
•Eosinophilia (>1.5 x 10ꝰ/L) lasting for more than 6 months without an identifiable cause
Eosinopenia
•Absolute Eosinophil Count of less than 0.09 x 10ꝰ/L
•Most often associated with conditions that result in marrow hypoplasia
•Absolute eosinophilia is associated in: Scarlet fever, HIV, fungal infections, Autoimmune disorders, and hypersensitivity to antibiotics & anti-seizure medications
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
BASOPHILS
•Basophilia is defined as an
absolute basophil count greater
than 0.15 x 10ꝰ/L
•Normal relative basophil count: 0-
2%
•Normal absolute basophil count:
0-0.2 x10^9?L
•Most common cause: presence
of malignant myeloproliferative
neoplasm such as CHRONIC
MYELOGENOUS LEUKEMIA (CML)
Note:
NONMALIGNANT CAUSES OF
BASOPHILIA
üAllergic rhinitis
üHypersensitivity to drugs
or food
üChronic infections
üHypothyroidism
üChronic inflammatory
conditions
üRadiation therapy
üBee stings
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
MONOCYTES
•Monocytosis is defined as an
absolute monocyte count >1.0 x 10ꝰ/L (adults) and > 3.5 x 10ꝰ/L
(neonates)
•Normal relative monocyte count:
2-11%
•Normal absolute monocyte
count:0.1 –1.3 x 10^9/L
•Monocytosis is frequently the FIRST
SIGN OF RECOVERY from acute
overwhelming infection or severe
neutropenia
Note:
MONOCYTOPENIA
•Absolute monocyte count of <0.2 x10ꝰ/L
•Associated in patients with:
üAplastic anemia
üChemotherapy-induced cytopenia
üPatients receiving steroid therapy
üHemodialysis patients
üSepsis
üEpstein-Barr virus (EBV)
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
LYMPHOCYTES
•Lymphocytosis is suspected in
patients with absolute lymphocyte count >10.0 x 10ꝰ/L
(children) and >4.5 x 10ꝰ/L (adults)
•Normal relative lymphocyte
count: 18-42%
•Normal absolute lymphocyte
count: 1.0-3.2x10^9/L
•Lymphocytopeniacan be
defined as an absolute
lymphocyte count <2.0x 10ꝰ/L
(children) and <1.0 x 10ꝰ/L (adults)
TRIKS PICAR, RMT

QUANTITATIVE ABNORMALITIES OF LEUKOCYTES
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
1. TOXIC GRANULATION
•Appears as dark, blue-black
granules in the cytoplasm of
neutrophils
•PEROXIDASE: (+)
•Positively correlated with levels of
CRP
•The intensity of toxic granulation is a
general measure of inflammation
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
2. DOHLE BODIES
•Cytoplasmic inclusions consisting of remnants of ribosomal RNA arranged in parallel rows
•Typically found in band and segmented neutrophils and often in cells containing toxic granulation
•Intracytoplasmic pale blue round or elongated bodies between 1um-5um in diameter
•Relatively non-specific
•Maybe present in: Bacterial infections, Sepsis and Normal pregnancy
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
3. CYTOPLASMIC VACUOLATION
•Vacuoles generally reflect
phagocytosis
•AUTOPHAGOCYTIC VACUOLES:
small (2um) and distributed
throughout the cytoplasm
•Can be induced by DRUGS such as:
Sulfonamides and Chloroquine,
storage of blood in EDTA for >2 hrs
hours, autoantibodies, acute
alcoholism and exposure to high
doses of radiation
•PHAGOCYTIC VACUOLES:
large (up to 6um) and are
frequently accompanied
by toxic granulation
•When seen, a careful
examination of the film
should be made for
presence of bacteria or
yeast (Ehrlichiaand
Anaplasma)
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
4. PYKNOTIC NUCLEI
•Generally indicate
IMMINENT CELL DEATH
•Nuclear water has been
lost and the chromatin
becomes very dense and
dark, but filaments can still
be seen between segments
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
5. NECROBIOTIC NUCLEI/CELL
•Found in dead cells
•Rounded fragments of
nucleus with NO
FILAMENTS and NO
CHROMATIN PATTERN
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
6. DEGRANULATION
•Common finding in activated
neutrophils and eosinophils
•Granules are released into the
extracellular space
•In vitro degranulation often occurs
when cell membranes of the cells
are disrupted during the process of
making blood films
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
NEUTROPHILS
7. CYTOPLASMIC SWELLING
•Caused by actual osmotic swelling
of the cytoplasm or by increased
adhesion to the glass slide by
stimulated neutrophils
•Results in variation in neutrophil size
or neutrophil anisocytosis
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
MONOCYTES
1.IMMATURE/REACTIVE MONOCYTES
•Maybe seen during: Infections,
Recovery from BM aplasia and after
GM-CSF Adminisitration
•Nucleus can become thin and
band-like and may appear to be
segmenting
•Large numbers are seen in
hematologic neoplasms involving
the monocytic series
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
LYMPHOCYTES
•Reactive changes in lymphocytes
are referred to as: variant
lymphocytes, reactive
lymphocytes, effector lymphocytes,
transformed lymphocytes, Turk cells,
Downey cells, immunoblasts, and
atypical lymphocytes
•Result of complex morphologic and
biochemical events that occur as
lymphocytes are stimulated when
interacting with antigens in
peripheral lymphoid organs
Note:
PLASMACYTOID LYMPHOCYTE
•Type of reactive
lymphocyte that has some
of the morphologic
features of plamacytes
INFECTIOUS MONONUCLEOSIS
•Causative agent: EBV
•The virus infects the B
lymphocytes attached to
CD21
TRIKS PICAR, RMT

MORPHOLOGIC CHANGES IN LEUKOCYTES
LYMPHOCYTES
TRIKS PICAR, RMT

LEUKEMIAS,
MYELODYSPLASITC SYNDROMES (MDS), &
MYELOPROLIFERATIVE DISORDERS (MPD)
TRIKS PICAR, RMT

Courageous Caitie
Case: Juvenile
Myelomonocytic Leukemia
(JMML), a rare cancer of the
blood affecting infants and
toddlersTRIKS PICAR, RMT

Leukemia
‘leukos’ and ‘heima’
(cancer-WBC)
High abnormal
WBC (immature)
Low RBC, platelets
LN
Spleen
Liver
CNS
TRIKS PICAR, RMT

CANCER
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Leukemia
§Duration of untreated disease
§Acute leukemia -rapidly progressive up to 6 months
§Subcutaneous leukemia-2-6 months
§Chronic leukemia-minimum of 1-2 years depending on the age , type
of cell involved.
§# of WBC present in the PBS
§Leukemic leukemia->15,000/uL
§Subleukemicleukemia-<15,000/uLwith immature and abnormal cells
§Aleukemicleukemia-<15,000/uLwith no immature and abnormal
cells.
§Type of WBC involved
§Acute leukemia-predominance of immature cells (blast, and pro
stages)
§Chronic leukemia-the cell types involve are the mature cells
§Myelocytic(myelogenous, myeloblastic, granulocytic)
§Lymphocytic (lymphogenous, lymphtic)
§Monocytic
§Plasmacyticleukemia, megakaryocytic and erythroid leukemia
TRIKS PICAR, RMT

TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

Acute vs Chronic Leukemia
AcuteChronic
AgeChildren & young
adults
Middle age and
elderly
OnsetSuddeninsidious
Durationweeks to monthsyears
WBC countVariable High
TRIKS PICAR, RMT

Acute vs Chronic Leukemia
AcuteChronic
PlateletsLowEarly: Normal/ High
Late: Low
AnemiaHigh (>90%)None/ mild
Predomi-
nantcells
Blast cellsMature cells
AML = myeloblast
ALL= lymphoblast
CML=granulocytes
CLL=lymphocytes
TRIKS PICAR, RMT

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

TRIKS PICAR, RMT

Acute Leukemia
TRIKS PICAR, RMT

TRUE or FALSE
•Acute lymphocytic leukemia (ALL) is the most common type
of leukemia in young children and it never affects adults.
TRIKS PICAR, RMT

AML vs CML
TRIKS PICAR, RMT

Acute vs Chronic Leukemia
TRIKS PICAR, RMT

ACUTE MYELOID
LEUKEMIA
§Hereditary-certain syndromes seen associated with AML such as Down syndrome, Fanconi’s syndrome, Bloom syndrome.
§Acquired-radiation; chemical-benzene; paint, embalming chemicals, pesticides, petroleum; personal activities-smoking
§Classification is categorized according to morphology, cytochemistry, and immunophenotype
TRIKS PICAR, RMT

Acute Myelogenous Leukemia (AML)
•acute nonlymphocytic 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)
TRIKS PICAR, RMT

TRUE or FALSE
•Acute myeloid leukemia (AML) is sometimes called acute
nonlymphocytic leukemia (ANLL).
TRIKS PICAR, RMT

TRIKS PICAR, RMT

AML (+) Bb. Thomson
TRIKS PICAR, RMT

Peripheral Blood Blast Cell
TRIKS PICAR, RMT

Normal bone marrow
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

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

Acute myeloid leukemias (AML)
Classification -FAB
M0: minimally differentiated
M1: myeloblastic leukemia without maturation
M2: myeloblastic leukemia with maturation
M3: hypergranularpromyelocytic leukemia
M3V-hypogranularpromyelocytic leukemia
M4: myelomonocytic leukemia
M4Eo: variant, increase in marrow eosinophils
M5A-monoblastic leukemia/ monocytic without maturation
M5B-monocytic with maturation
M6: erythroleukemia (DiGuglielmo'sdisease)
M7: megakaryoblasticleukemia
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

M5
M4
M1
M2
M3
M7
MEGAKARYOCYTIC
CELLS
M0
MYELOBLASTMONOBLASTERYTHROID
CELLS
M6
MYELOID
CELLS
STEM CELLS
PROMYELOCYTE
M4E0M5aM5b
M6
TRIKS PICAR, RMT

TRIKS PICAR, RMT

AML
TRIKS PICAR, RMT

Acute Nonlymphocytic Leukemias
TRIKS PICAR, RMT

CYTOCHEMICAL STAINS
STAINPURPOSE SPECIMEN
Leukocyte
alkaline
phosphatase
stain
Stains ALPpresent in the neutrophil,
and to a small degree, in certain B
lymphocytes,
Helpful in differentiating chronic
myelogenousfrom a leukomoidreaction
or polycythemia vera.
Fresh capillary blood is
recommended.
Alternatively, bloodmay be
collected using heparin as
anticoagulant. (EDTA has an
inhibitory effect on the stain
reaction and will yield a falsely
low result)
PeroxidaseStains peroxidsespresent in the
granulocytesand monocytes.
Used to differentiate acute myelogenous
and monocyticleukemiasfrom acute
lymphocytic leukemia
Fresh blood smears made from
capillary blood are recommended,
or use fresh whole blood
anticoagulatedwithEDTAor
heparin)
Sudan Black BStains lipids present in the monocytes
and granulocytes.
Used to differentiate acute myelogenous
and myelomonocyticleukemiasfrom
acute lymphocticleukemia
Air dried bone marrow smears
TRIKS PICAR, RMT

CYTOCHEMICAL STAINS
STAINPURPOSESPECIMEN
Chloroacetate
esterase
Stains esterasespresent in
the granulocytes.
Used to differentiate
granulocytic cells from
monocyticcells.
Air dried blood or bone
marrow smears.
Blood anticoagulated
with EDTA or heparin
may also be used.
Nonspecific
esterase
Stains esterasespresent in
the monocyticcells,
macrophages ,
megakaryocytes and
platelets.
Used to differentiate
monocyticleukemiasfrom
granulocytic leukemias
Air dried blood or bone
marrow smears.
Blood anticoagulated
with EDTA or heparin
may also be used.
TRIKS PICAR, RMT

CYTOCHEMICAL STAINS
STAINPURPOSESPECIMEN
Periodic acid –
Schiff
Stains mucoproteins, glycoproteins and
high molecular weight carbohydrates
normally present in almost all blood cell
type except pronormoblast.
Used to help in diagnosis of
DuGuglielmo’s syndrome and may be an
aid, when used in conjuction with other
stains, to classify some acute leukemias
Air dried blood or
bone marrow smears
Acid PhosphataseStains ACP present in the myelogenous
cells, lymphocytes, plasma cells, monocytes
and platelets.
Using L(+) tartaric acid, the stain is helpful
in diagnosing hairy cell leukemia
Air dried blood or
bone marrow smears,
or use blood
anticoagulated with
heparin
TRIKS PICAR, RMT

SPECIAL STAINS
NAMETYPECONSTITUENT
S STAINED
INTERPRETATIO
N
IMPORTANT
INFORMATIO
N
Myeloperoxidas
e (MPO)
EnzymeMarker for
primary granules
and Auer rods
Peroxidase activity
produces dark
browngranules in
cytoplasm of
granulocytes and
monocytes
Myeloperoxidas
e enzyme
deteriorates.
Stains should
be done only on
fresh specimens
SudanBlack B
(SBB)
Non
enzyme
Markerfor
phospholipids
and lipids
Dark purple-black
granules in
neutrophil
precursors.
Lymphoblastsare
negative
Canbe done on
stored
specimens.
Parallels MPO
for
interpretation
Terminal
Deoxyribonucle
otidyl
Transferase
(TdT)
EnzymeDNA polymerase
immunoperoxidas
e
Is present in 90%
cases of ALL
Usedto
differebtial
AML to ALL
TRIKS PICAR, RMT

SPECIAL STAINS
NAMETYPECONSTITUENTS
STAINED
INTERPRETATIONIMPORTANT
INFORMATION
PeriodicAcid-SchiffNonenzym
e
Markerfor glycogen,
glycoproteins,
mucoproteinsand high
molecular weight
carbohydrates
Activity results in
bright fuschiapink
Pattern of staining
varies with each cell
type
Lymphoblastic
leukemia shows blocky
or chunky pattern
L1 and L2 block pattern
L3 negative
Erythroblast in M6
leukemia positive
NaphtolAS-D
Chloroacetate
esterase
Enzyme Marker for mature and
immature neutrophils
and mast cells
Enzyme activity
results in bright red
granules in cytoplasm
of neutrophils,
neutrophil precursors
and mast cells
Known as specific
esterase
Stable enzyme that
lasts for months
a-naphthylAcetate
Esterase
EnzymeMarker for monocytes,
megakaryocytes, and
plasma cells
Monocytes stain red-
brown
Known as non specific
esterase (NSE)
a-naphthylButyrate
Esterase
EnzymeIdentifying monocytes,
promonocytesand
monoblasts
Enzyme activity
results in dark red
precipitates in
cytoplasm
Known as non specific
esterase (NSE)
Tartrate Resistant
Acid Phosphatase
EnzymeMarkerfor hairy cell
leukemia
Activity is indicated
by purple to dark red
granules in cytoplasm
Excellent marker for
hairy cell leukemia
Acid phosphatase EnzymePresent in all
hematopoietic cells are
found in lysosomes
Activity is indicated
by purple to res
granules
Cannot be storedTRIKS PICAR, RMT

Special stains
NAMETYPECONSTITUEN
TS STAINED
INTERPRETA
TION
IMPORTAN
T
INFORMAT
ION
Leukocyte
Alkaline
Phosphatase
(LAP)
EnzymeNeutrophilis the
only leukocyte
that contains
this activity
100 cell count is
done.
Neutrophilsare
scored form with
no activity to 4
with a large
amount of
activity
Useful to
differentiate
CML from a
leukomoid
reaction.
CML has
decreased
activity
Toluidine
Blue
Nonenz
yme
Binds with acid
mucopolysacchar
idesin blood
cells
Strongly
metachromatic
Useful for
recognition of
mastcells
and tissue
basophilsTRIKS PICAR, RMT

TypePASSBBMPOCAENSEAuerrods
M0-----Rare
M1-+++-50%
M2-+++-50%
M3-+++-95%
M4-++++64%
M5---_+0
M6+++--50%
M7+---+/_Rare
CytochemicalStains in Myeloblasts
TRIKS PICAR, RMT

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

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

MO: Minimally differentiated
•Undifferentiated Blasts (No
maturation)
•Myeloid phenotype -CD13,
CD33, CD34
•(-) SBB, MPO
•Negative: Auer rods, Esterase
TRIKS PICAR, RMT

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

M1 AML without maturation
TRIKS PICAR, RMT

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

M2 AML with maturation
TRIKS PICAR, RMT

Auer Rods
TRIKS PICAR, RMT

M3 (hypergranular promyelocytic)
•Promyelocyte-predominant
•Large, kidney shape
•(+) Auer rods (faggot cells)
•basophilic, bilobed nuclei
•CD 13,33
•High incidence of DIC
TRIKS PICAR, RMT

Acute myeloid leukemia with very abnormal
cells (AML M3/ t15;17)
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

M5: acute monocytic leukemia
1.M5a –without maturation
•Monoblasts, few promonocytes
2.M5b –with maturation
•Blast, Promonocytes (BM), Monocytes(Blood)
TRIKS PICAR, RMT

TRIKS PICAR, RMT

M5a
•Monoblast ameboid with
round to oval nuclei,
•prominent nucleoli,
•<20% promonocytes/mono
•Vacuolated cytoplasm
TRIKS PICAR, RMT

AML M5a
TRIKS PICAR, RMT

M5b
•> 20% promonocytes, monocytes
•Promonocytes folded,
convulated nucleus
•Azurophilic granules
TRIKS PICAR, RMT

AML M5b
TRIKS PICAR, RMT

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

M6 (erythroblast)
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

M7 (Megakaryoblast)
TRIKS PICAR, RMT

Megakaryoblast
TRIKS PICAR, RMT

Acute lymphocytic
leukemia (children)
TRIKS PICAR, RMT

TRIKS PICAR, RMT

Acute Lymphocytic Leukemia
French-American-British (FAB)
Classification
L1ALL, child
L2ALL, adult
L3 ALL, (Burkitt)
TRIKS PICAR, RMT

WHO classification-Cytogenetics
Chromosomal
Abnormality
t(1;19)
t(11;14)
t(8;14), t(2;8), t(8;22)
Leukemia
ALL-pre B
ALL-T
ALL-Burkitt’slymphoma
TRIKS PICAR, RMT

Immunophenotyping
TypesFABTdTTcAgBcAg
Precursor BL1,L2+, --+
Precursor TL1,L2+, ++-
B-cell L3-,--+
TRIKS PICAR, RMT

Acute Lymphocytic
Leukemia (ALL)
L1
small uniform cells
(-) nucleoli
L2
large varied
(heterogenous)
cells, (+) nucleoli
L3
large varied cells vacuoles
(bubble-like)
TRIKS PICAR, RMT

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 (+)
TRIKS PICAR, RMT

Acute Lymphocytic Leukemia FAB Class
L2
•Large, heterogenous
•basophilic cytoplasm
•Nucleoli present
•50% c-ALL
30% T-ALL
•TdT (+)
•PAS (+)
TRIKS PICAR, RMT

Acute Lymphocytic Leukemia FAB Class
L3 (Burkitt’s)
•large varied cells
•deeply basophilic
•vacuolated cytoplasm
•Nucleoli (+)
•Usually B-ALL
•TdT (-)
•PAS (+)
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

CHRONIC
LEUKEMIAS
TRIKS PICAR, RMT

TRIKS PICAR, RMT

Chronic Myeloproliferative
Diseases (MPD)
TRIKS PICAR, RMT

Classification of chronic myeloproliferative
diseases (MPD)
FAB WHO
1.Chronic myelogenous
leukemia (CML)
CML Ph+: t(9;22)(qq34;q11),
BCR/ABL
Chronic neutrophilicleukemia
2. Polycythemiavera(PV) Polycythemiavera(PV)
3. Essential thrombocytemia(ET) Essential thrombocytemia(ET)
4. Chronic MyelofibrosisChronic idiopathic
myelofibrosis
TRIKS PICAR, RMT

Myeloproliferative disease (MPD)
DisorderCell TypeFeature
CMLGranulocytes(Ph1), LAP/NAP, Splenomegaly
PVErythrocytesRBC, Splenomeg, Teardrop,
High LAP
ETPlateletsThrombocytosis
CMFFibroblastNRBC, LAP, Dacrocytes
TRIKS PICAR, RMT

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)TRIKS PICAR, RMT

Chronic Myeloid Leukemia
TRIKS PICAR, RMT

Leukemoid
reaction
CML
WBCHighHigh
Anemia(-)(+)
PBSShift to the Left
Toxic granulation
Dohlebodies
Shift to the left (blast)
Eosinophilia,
basophilia
LAP scoreHighLow
Philadelphia
chromosome
(-)(+)
TRIKS PICAR, RMT

Chromosomal
Abnormality
Leukemia
t(9;22) -Philadelphia
chromosome
CML
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Polycythemia vera (PV)
•Malignant hyperplasia of the multipotential myeloid stem
cell causes increase in all cell lines (polycythemia);
erythrocytes most greatly increased despite decreased
erythropoietin (EPO); inappropriate erythropoiesis
•High blood viscosity can cause high blood pressure, stroke,
and heart attack.
•Found in adults 50 years of age and older
TRIKS PICAR, RMT

Polycythemia vera (PV)
•Laboratory: Increased RBC (7-10 X 1012/L), hemoglobin (>20
g/dL), and hematocrit (>60%) along with increased
leukocytes and platelets indicate polycythemia. RBC mass is
increased with a normal plasma volume.
•Treatment is therapeutic phlebotomy, splenectomy, and
chemotherapy. PV is a chronic disease with a life expectancy
after diagnosis of up to 20 years.
TRIKS PICAR, RMT

Polycythemia vera (PV)
•Must differentiate from other forms of polycythemia
1) Secondary polycythemia
a) Increase in RBC mass is an appropriate response to
increased EPO or tissue hypoxia. Plasma volume, leukocyte
count, and platelet count are normal.
b) Can be caused by smoking, emphysema, or high altitude
TRIKS PICAR, RMT

Polycythemia vera (PV)
2) Relative (pseudo-) polycythemia
a) Decreased plasma volume with a normal RBC mass caused
by dehydration (diarrhea, diuretics, or burns)
b) Increased hemoglobin, normal leukocyte and platelet count,
normal EPO
TRIKS PICAR, RMT

MYELOFIBROSIS with Myeloid
Metaplasia
uMF –char by anemia, abnormal proliferation of all
myelocytictissue and fibrosis of Bone Marrow.
uMM –extramedullaryorgans (liver and spleen)
becomes hematopoietic and resemble the histologic pattern of BM
uAlso known as myelosclerosiswith MM or idiopathic
myeloid metaplasia
uMiddle-aged & elderly (58 y/o)
uCause-Unknown (Radiation and benzene poisoning ) -
inflammation of the BM
*Many cases among the survivors of Hiroshima bombing and workers in
rubber industry. TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Essential Thrombocythemia
•Proliferation megakaryocytes (adults)
•High platelet count, giant form
•Platelet function abnormalities
•Usually without hepatomegaly and splenomegaly
•Found mainly in adults 60 years of age and older
•Laboratory: Platelets commonly greater than 1000 X 109/L,
giant forms, platelet function abnormalities, leukocytosis
TRIKS PICAR, RMT

Myelodysplastic
Syndromes(MDS)
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

Refractory anemia (RA)
-Anemia that is refractory (not responsive) to therapy
-Laboratory: Oval macrocytes, reticulocytopenia,
dyserythropoiesis; bone marrow blasts <5% and peripheral
blood blasts <1%
TRIKS PICAR, RMT

Refractory anemia with ringed
sideroblasts (RARS)
•Ringed sideroblasts comprise more than 15% of bone marrow
nucleated cells. Signs of dyserythropoiesis, neutropenia
•Laboratory: Similar to RA; dimorphic erythrocytes
•This is the primary/idiopathic sideroblastic anemia discussed
with the anemias
TRIKS PICAR, RMT

Chronic myelomonocytic leukemia
(CMML)
•The one MDS that usually presents with leukocytosis
•Laboratory: Bone marrow blasts 5-20% and peripheral blood
blasts <5%; absolute monocytosis greater than 1.0 X 109/L
TRIKS PICAR, RMT

Refractory anemia with excess blasts
(RAEB)
•Trilineage cytopenias common
•Laboratory: Bone marrow and peripheral blood blasts are
the same as with CMML, but there is no absolute
monocytosis.
•The higher the blast percent, the worse the prognosis.
TRIKS PICAR, RMT

Refractory anemia with excess
blasts in transformation (RAEB-t)
•Laboratory: bone marrow blasts >20% but less than 30%;
peripheral blood blasts >5%
•WHO classification reassigns RAEB-t as an acute leukemia
instead of a myelodysplastic syndrome because of the bone
marrow blast percent.
TRIKS PICAR, RMT

•WHO classification of MDS has additional groups (e.g.,
refractory cytopenia with multilineage dysplasia, 5q deletion
syndrome).
•WHO created the new category of
myelodysplastic/myeloproliferative disease, which includes
the FAB's CMML.
TRIKS PICAR, RMT

Myelodysplastic
syndromes (MDS)
(FAB)
RARARSRAEB
(6-20%
myeloblast
RAEB-T
( 21-30%
myeloblast
< 5% myeloblast> 5% myeloblast
chronic myelomonocytic leukemia
(CMML) = 20% myeloblast
monocytosis
splenomegaly
TRIKS PICAR, RMT

TRIKS PICAR, RMT

RARS
>15% ringed sideroblast (PB)
TRIKS PICAR, RMT

RAEB-T (vacuolated blast)
•>20% marrow blasts
•<30% peripheral blast
•WHO-acute leukemia instead
MDS
•FAB -CMML
TRIKS PICAR, RMT

RAEB-T
granular & clot likevacuolationTRIKS PICAR, RMT

LYMPHOID
LEUKEMIAS
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Chronic Lymphocytic Leukemia (CLL)
TRIKS PICAR, RMT

Hairy Cell Leukemia (HCL)
•B cell malignancy (CD 19,
CD20)
•Marked splenomegaly
•Pancytopenia
•Cytoplasm show hair like
projection
•(+) TRAP
TRIKS PICAR, RMT

Hairy Cell Leukemia (HCL)
TRIKS PICAR, RMT

Prolymphocytic Leukemia
•B cell or T cell malignancy
•Marked splenomegaly
•Lymphocytosis, prolymphocytes
•Anemia, thrombocytpenia
TRIKS PICAR, RMT

Plasma Cell Dyscrasia
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Multiple myeloma
TRIKS PICAR, RMT

Normal BM / MM in BM
TRIKS PICAR, RMT

Plasma Cell Leukemia
•Abnormal plasma cells in blood
•Pancytopenia
•Rouleaux
•Monoclonal gammopathy
TRIKS PICAR, RMT

Plasma cell Disorder•Waldenstrom’s macroglobulinemia-disease of the elderly,
presenting with weight loss and fatigue. It is a low grade lymphoma like disease characterized by infiltration of the bone
marrow with small but mature B lymphocytes.
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Lymphomas
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

Non Hodgkin
Lymphoma
§Lowgrademalignancy
§Malignantlymphoma,Smalllymphocytic(SL)
§Malignantlymphoma,Follicular,PredominantlySmallCleavedCells
(FSC)
§MalignantLymphoma,Follicular,MixedSmallCleavedandlargecells
(FM)
§Intermediategrademalignancy
§Malignantlymphoma,Follicular,PredominantlyLargecells(FL)
§Malignantlymphoma,DiffusedSmallCleavedcell(DSC)
§Malignantlymphoma,DiffuseSmallandlargeCleavedCell(DM)
§Malignantlymphoma,DiffuseLargeCell(DL)
§HighGrademalignancy
§Malignantlymphoma,LargeCellImmunoblast(IBL)
§Malignantlymphoma,Lymphoblast(LBL)
§Malignantlymphoma,SmallNoncleavedCells(SNC)
TRIKS PICAR, RMT

Hodgkin Lymphoma
•EBV associated
•(+) Reed Strenberg
cells = large
multinucleated cells
with large nucleoli
•Mild anemia,
eosinophilia,
monocytosis
•High LAP, ESR
TRIKS PICAR, RMT

Hodgkin’s
Lymphoma
§Lymphocytic predominance –usually
diffused, vaguely nodular in pattern,
abundant lymphocytes, few reed
Sternberg cells of fibrosis
§Nodular sclerosis-nodular pattern
formed by bifringent collagen bands,
mod number of lymphocytes,
eosinophils , plasma cells, lacunar
variant of reed Sternberg cells
§Mixed cellularity-diffuse involvement,
numerous reed Sternberg cells,
moderate number of lymphocytes,
eosinophils, and plasma cells
§Lymphocyte depletion-diffuse
involvement, decrease cellularity,
occasional numerous bizarre shaped
reed Sternberg cells
TRIKS PICAR, RMT

Other Lymphoma
§Sezary syndrome-malignant lymphoma affecting the skin and
involves primarily the T lymphocytes. Positive for monoclonal antibodies for CD3, CD2 and CD4. Sezary cells are present –
medium sized lymphocyte with convoluted nucleus.
Mycosis fungoides -is the most common form ofcutaneous T-cell
lymphoma. It generally affects the skin, but may progress internally over
time. Symptoms include rash, tumors, skin lesions, and itchy skin.TRIKS PICAR, RMT

Other Lymphoma
´Burkitt’s syndrome-most often found in children in Africa and New Guinea, affects the facial bone and jaw. EBV is involve in transforming the morphology of B lymphocytes
TRIKS PICAR, RMT

DIAGNOSTIC
PROCEDURES
TRIKS PICAR, RMT

TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

Leukemia Diagnosis
7. Blood chemistry
•ALP = CML diagnosis
•Vitamin B12= CML
•Ca, K, phosphate, uric acid = ALL
TRIKS PICAR, RMT

TRIKS PICAR, RMT

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

Cytomorphology
NSE
Pappenheims
MPO
TRIKS PICAR, RMT

Cytochemical stain
1.Myeloperoxidase (MPO) stain
•(+) AML (granulocyte, monocyte, Auer rods)
•(-) ALL
2.Sudan Black B ( phospholipids, proteins)
•(+) AML (granulocyte, monocyte, Auer rods)
•(-) ALL
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

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

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
TRIKS PICAR, RMT

Immunophenotyping
TypesFABTdTTc AgBc Ag
c Ag s Ag
Precursor
B
L1,L2+, --+-/+-
Precursor TL1,L2+, ++---
B-cell L3-,--++-/+
TdT = Terminal deoxynucleotidyl transferase
CAg= common Ag
Sag= surface antigen TRIKS PICAR, RMT

CD markers
•B cell= C19, CD20
•T cell = CD2, CD3, CD5
•Myeloid= CD13, CD14, CD33
CD or Cluster of Differentiation =is a protocol used for the
identification and investigation ofcell surface
moleculesproviding targets forimmunophenotypingof cells.
TRIKS PICAR, RMT

3. Multiparameter flow
cytometry
EDTA heparinized BM or blood
Cell lysis/ Ficoll hypaque gradiation
MFC
TRIKS PICAR, RMT

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
TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

Cytogenetics
•Metaphase after G-banding in a
c-ALL with t(9;22) so called
Philadelphia translocation
TRIKS PICAR, RMT

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)
TRIKS PICAR, RMT

Metaphase before and after analysis by 24-color FISH in a case
with AML and complex aberrant karyotype
TRIKS PICAR, RMT

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

end na po…
TRIKS PICAR, RMT

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