Fig. 1.

Enzymatic cytochemistry of blood cells of fat snook: (a)

stain for myeloperoxidase on neutrophil; (b e f) stain for acid
definiti
phosphatase on Young neutrophil, mature neutrophil,
lymphocyte, monocyte, and thrombocyte, respectively; (gon e j)
stain for non-speci fi c esterase on neutrophil, lymphocyte,
monocyte and thrombocyte, respectively. Bar 14 5 m m.
-Uncontrolled cell proliferation: too
many cells

-Abnormal function: cells dont work

-Immunosuppression:
severe, life threatening
infections

-Imbalanced hemostasis:
Leukemia - outcomes
hemorrhage

-Decreased oxygen carrying

capacity: anemia, tissue
death, organ failure

-Acute: rapid, progressively

worsening clinical course

Time of Onset (Classifications)

-Chronic: indolent clinical

course

-Acute: proliferation of
immature cells: arrested
Cell maturation (Classifications) maturation
-Cancer (Malignancy) of blood and
blood forming tissues
 -Anemia
-Chronic: proliferation of
mature looking cells: normal -Peroxidase in granules in
maturation myelocytic and monocytic
cell lines
Peroxidase - Myeloperoxidase
-Anemia
(MPO) - principle
-Most strongly in myelocytic
-Thrombocytopenia: cells
bleeding
-Peroxidase + H2O2
-Neutropenia: infections oxidizes a dye substrate
creating a black/blue/brown
-Bone pain: expansion of ppt
marrow
-Differentiates AML vs ALL
-Weightloss
Leukemia - symptoms
-Evaluate blasts only
-Hepatosplenomegaly

-Lymphadenopathy

-Rashes/tumors/CNS -Stains Primary

involvement tissue
infiltrates of neoplastic cells
(chloromas or myeloid Granules
tumor) -neutrophils

Myeloperoxidase - interpretation
-Decr to mod incr WBC -eosinophils

-N to decr PLTs -monocytes

Acute leukemia - lab results
-Abnormal morphology:
Large, hypogranular,
micromegakaryocytes
-stain = Acute Myeloid

-Incr to markedly incr WBC Leukemias

Chronic leukemia - lab results

-N to incr PLTs -no stain = Acute Lymphoid

 Leukemias

-AML will see incr staining of

myelocytic and monocytic
blasts
MPO stain smear

MPO stain smear

Sudan Black B smear

-Stains lipids such as

sterols, neutral fats &
phospholipids

Sudan Black B smear

Sudan Black B (SBB) - principle

-found in granules of
neutrophils & monocytes

-hydrolyze an ester
substrate

-Differentiates AML vs ALL

Esterases - principle

-Consider blasts only

Sudan Black B (SBB) -
interpretation -A napthol compound is
released and combines with

Specific Esterase - general
Nonspecific Esterase (NSE) -
general
Combined Esterase - general
Periodic Acid Schiff (PAS) -
principle
a diazonium salt that ppts
-napthol AS-D chloroacetate
-granulocyte esterase
-myelocytic blasts will
stain
---primary granules
---auer rods
-Alpha-napthyl acetate or
alpha-napthyl butyrate
-Monocytic
cells are strongly pos, & in
hibited w/ NaF
-Myelocytic cells may
be weakly pos, &
remains weakly pos w/ NaF
-Combines specific and
nonspecific in one dye (Not
commonly used)
-Acid oxidizes glycogen,
mucoproteins & other high
molecular wt. carbs to
aldehydes
-Aldehydes react w/ Schiff
rgt staining bright pink
-Positive rxn in malignant
lymphocytic &
erythrocytic cells (chunky
or blocky)
-Negative in benign cells
-Megakaryocytes will also
stain, but diffuse
-In secondary granules of
neutrophils
-Substrate: napthol AS-BI
phosphate is hydrolyzed by
Leukocyte Alkaline Phosphatase
LAP
(LAP) - principle
-Hydrolyzed substrate
combines w/ a dye &
precipitates (color depends
on substrate used)
-Score 100 PMNs and
LAP - interpretation
Bands on a scale of 0 - 4+
based on the amount of ppt
in the cell

LAP reactions

-Used to Investigate a shift

to the left

-present in almost all

nonerythroid cells
-Malignant granulocytes
(CML): Decr LAP vs.
leukomoid rxn (incr LAP)

-hydrolyzes substrate
napthol AS-BI phosphoric
acid
-Other causes of decreased
LAP: PNH, Sideroblastic
anemia, Myeloproliferative
disorders

-Hydrolyzed substrate
Tartrate Resistant Acid
couples w/ a dye to form a
Phosphatase (TRAP) - principle
red ppt

-CML will have REDUCED

score

LAP scale
-All normal isoenzymes of
the enzyme are inactivated
by tartaric acid and will not
stain.

-In Hairy Cell Leukemia

 isoenzyme #5 is resistant evaluated. of cells
to tartaric acid and will 3. Statethe substance 3. Black/blue/brown
stain the stain identifies precipitate stain
(ex: glycogen) and 4. AML positive

-A specific cell marker the cell (myeloblast or

(enzyme) that catalyzes the stainingpatterns. monoblast)

polymerization of (which cells are

deoxynucleotides found only positive and which

in lymphocytic precursors. are negative)

Absent in lymphocytes 4. Interpret the results

of the stainslisted.

Terminal deoxyribonucleotidal
Transferase (TdT) - principle 1. Sudan Black B
(SBB)
2. Name the cell
-Not a cytochemical stain: is stage(s) that is 1. myeloblasts and
an immunofluorescent stain evaluated. monoblasts
(flow cytometry) or 3. State the 2. Stains lipids, neutral
immunoperoxidase substance the stain fats, and phospholipids
technique identifies (ex: found in neutrophils and
glycogen) and the monocytes.

-Positive in acute cell staining 3. black staining granules

lymphocytic leukemias ( L1 patterns. (which 4. AML positive

& L2 ) cells are positive (myeloblasts and

and which are monoblasts)
negative)
TdT - interpretation
4. Interpret the results
of the stains listed.

-Must interpret carefully: TdT 1. Specific esterase

has been observed in AML (Napthol AS-D
Choroacetate
1. myeloblasts
esterase)
2. identifies granulocyte
2. Name the cell
s the purpose of esterase
1. To differentiate stage(s) that is
ming cytochemical 3. bright red stain across
types of acute evaluated.
? the cell indicate positive
leukemia from one 3. State the
reaction
another. substance the stain
4. positive indicates AML,
identifies (ex:
myeloblasts only
1. Myeloperoxidase 1. myeloblasts and glycogen) and the

2. Namethe cell monoblasts cell staining

stage(s) that is 2. peroxidase in granules patterns. (which

 cells are positive negative) (diffuse)
and which are 3. Interpret the results
negative) of the stains listed.
4. Interpret the results
of the stains listed. Terminal
deoxynucleotidal
1. Nonspecific transferase (TdT)
esterase (NSE)
(alpha-napthyl
acetate esterase)
2. Name the cell
1. lymphoblasts
stage(s) that is 1. monoblasts 1. Name the cell
2. Immuno-fluorescent
evaluated. 2. Positive stain is black stage(s) that is
stain that catalyzes the
3. State the precipitate evaluated.
polymerization of
substance the stain 3. Monoblasts stain black. 2. State the
deoxynucleotides found
identifies (ex: 4. If NaF (sodium fluoride) substance the stain
in lymphocytic
glycogen) and the is added to NSE stain identifies (ex:
precursors only.
cell staining the monoblasts will not glycogen) and the
3. Positive cells fluoresce
patterns. (which stain. cell staining
4. Positive in ALL (L1 and
cells are positive patterns. (which
L2).
and which are cells are positive
negative) and which are
4. Interpret the results negative)
of the stains listed. 3. Interpret the results
of the stains listed.

Periodic acid schiff

(PAS) Leukocyte alkaline
1. Lymphoblast, phosphatase (LAP)
Rubriblast, 1. Stain identifies
Megakeryoblast secondary granules of
2. PAS oxidizes glycogen, neutrophils. LAP
1. Name the cell mucoprotein, HMW hydrolyzes napthol AS-
stage(s) that is carbs to aldehydes 1. Name the cell BI phosphate which
evaluated. which stain a bright pink stage(s) that is combines with a dye
2. State the color. evaluated. and changes color.
substance the stain 3. positive result is fine 2. State the 2. 100 PMNs are scored
identifies (ex: diffuse, coarse/granular substance the stain 0-4 based on amount of
glycogen) and the pink identifies (ex: precipitate.
cell staining 4. ALL (malignant glycogen) and the 3. increased LAP activity
patterns. (which lymphoblasts), cell staining seen in leukamoid rxn,
cells are positive malignant RBC blasts, patterns. (which polycythemia vera, and
and which are megakaryocytes cells are positive idiopathic myelofibrosis.
 and which are 4. decreased LAP in CML, LAP scores are 1. Leukamoid reaction -
negative) sideroblastic anemia, increased and 150 - 365; also CML of
3. Interpret the results myeloproliferative decreased? childhood which is
of the stains listed. disorder Ph^22 neg, 3rd
trimester pregnancy,

Acid polycythemia vera.

phosphatase/tartrat 2. Normal - 15 - 70

e resistant acid 3. CML (malignant

phosphatase granulocytes) - 0 - 24;

(TRAP) also PNH, sideroblastic

anemia,
1. Hairy cell leukemia
myeloproliferative
isoenzyme #5
disorders.
2. Red precipitate due to
acid phosphatase
1. Name the cell 1. Cancer (malignancy) of
hydrolysis with naphthol
stage(s) that is blood and blood
AS-BI.
evaluated. forming tissues.
3. Hairy cells are TRAP
2. State the 2. Uncontrolled cell
cells because they are
substance the stain proliferation, or
resistant to tartrate
identifies (ex: 1. What is leukemia? abnormal cell function
allowing them to
glycogen) and the 2. Why does it occur? 3. Immune suppression
hydrolyze with acid
cell staining 3. What conditions (acquired infections),
phosphatase.
patterns. (which does leukemia lead imbalanced hemostasis
cells are positive to? (bleeding), decreased
and which are O2 carrying capacity
negative) (anemia, tissue death,
3. Interpret the results organ failure).
of the stains listed.

1. acute leukemia has a

1. Scored 0 - 4+ based on rapid, progressively
the amount of worsening clinical
What differences
precipitate in the PMN. course, and shows
occur between
How do you 2. Count 100 PMN "arrested maturation" of
acute and chronic
determine the LAP 3. Multiply score (ex: 3+) cells.
leukemia in time of
for an appropriate by number of cells that 2. chronic leukemia has
onset and cell
cell? received that score (ex: an idolent clinical
maturation?
20). course, where "normal
4. Add the total score maturation" of cells is
together. seen.

What are the What are general 1. anemia

conditions in which clinical findings in 2. thrombocytopenia -
 leukemia? bleeding as 5% blasts
3. neutropenia - infections that are (+)
4. bone pain
5. weight loss
6. hepatosplenomegaly
7. lymphadenopathy
8. Rashes, tumors, CNS
involvement Look at BLASTS
ONLY

1. decreased to
moderately increased Stains lipids-
What are acute WBC sterols, neutral
leukemia lab 2. Normal to decreased fats, phospholipids
findings? PLT with abnormal
morphology
Sudan Black B
3. anemia
(SBB) principle

Found in granules

What are chronic 1. Increased to markedly of neutrophils and

leukemia lab increased WBC monocytes

findings? 2. Normal to increased

PLT AML (+) ALL(=)
3. Anemia

AML (+) ALL(=)

Primary and
secondary
granules of
SBB is positive
Stains primary neutrophils
in what
granules of
peroxidase is positive in leukemias?
neutrophils,
eukemias?
eosinophils, Also auer rods

monocytes

Lysosomal
granules of
monocytes

Can be as little
 butyrate

Consider BLASTS
ONLY

Esterases Monocytic cells are

hydrolyze an ester strongly (+) and

substrate inhibited by NaF

Esterases
principle
A napthol Myelocytic cells are

compound is (+/-) and remain so

released and with NaF

combines with a
diazonium salt that
precipitates

Napthol AS-D
chloroacetate

Myeloid leukemias-
primary granules
and auer rods
Specific
Specific
esterase positive
esterase Granulocyte
in what
compund esterase
leukemias?

BLASTS ONLY

Strong (+) in
monocytic line-
Nonspecific inhibited with NaF

Alpha-napthyl esterase positive

Nonspecific acetate in what

esterase leukemias?

compounds
Alpha-napthyl
 Weak (+/-) in
myelocytic

Negative in
BENIGN cells

BLASTS ONLY

Periodic acid
oxidizes glycogen, Can be fine/diffuse,

mucoproteins, and course/granular, or

other high MW mixture

carbohydrates to
aldehydes

Megakaryocytic
Periodic Acid blasts have strong
Schiff (PAS) positive PAS but
principle Aldehydes react diffuse
with Schiff reagent
staining bright
What cells can
PINK
have a diffuse
PAS pattern?

Neoplastic
myeloblasts may
show diffuse
pattern but not true
positive

Malignant
lymphocytic and Diagnose acute
erythrocytic cells and chronic
leukemias

PAS positive in
PAS stain
what leukemias?
details?

M6 and some ALLs

Looking for chunky
or block
 appearance substrate used)

Used to investigate
shift to the left

ALL can Score 100 PMNS

demonstrate a and bands on a
varied staining scale of 0-4+
pattern-some are based on amount
dififfuse of precipitate in cell
How to interpret
LAP stain

Some Normal is 15-70

lymphoblasts of
ALL may be PAS Leukomoid
negative reactions
(infections) 150-
Enzyme found in 365
secondary
When is LAP
granules of
increased?
neutrophils Some chronic
myeloproliferative
disorders like PCV,
idiopathic
myelofibrosis
Substrate napthol
Leukocyte
AS-BI phosphate is Chronic
Alkaline
hydrolyzed by LAP Myelogenous
Phosphatase
Leukemia 0-24
(LAP) principle

When is LAP *CML of childhood

The hydrolyzed decreased? (PH22 neg) usually
substrate high LAP
combines with a
dye and
precipitates (color PNH
depends on
 will not stain

Sideroblastic
anemia Hairy Cell
Leukemia-
What is TRAP
isoenzyme #5 is
used to
resistant to tartric
diagnose?
acid inhibition and
will stain
Myeloproliferative
disorders Specific cell marker
(enzyme) that
Acid phosphatase catalyzes the
is present in almost polymerization of
all non erythroid deoxynucleotides
cells found only in
LYMPHOCYTIC
PRECURSORS,
absent in
Terminal lymphocytes
deoxyribonucleot
Acid phosphatase idal Transferase
hydrolyzes (TdT) principle
substrate napthol
Tartrate AS-BI phosphoric
Resistant Acid acid. They NOT a
Phosphatase hydrolyzed cytochemical stain-
(TRAP) principle substrate couples is an
with a dye to form immunofluorescent
a ppt. stain (flow) or
immunoperoxidase
technique

Positive in acute
lymphocytic
All normal leukemias (L1 and
TdT
isoenzymes of acid L2)
interpretation
phosphatase are
inactivated by
tartaric acid and
 granules of
Neutrophils
Has been and
observed in AML Granulocytes.

Esterases
hydrolyzes an
ester
substrate. A
Normally present in naphthol
T-cells, pre-B cells, Esterases principle compound is
and a few BM cells realeased and
combines with
Peroxidase found in granules of a diazonium
dase cells in myelocytic and monocytic salt that
operoxidase cell lines plus H202 oxidizes a dye precipitates.
) Principle substrate creating a black-blue-
brown precipitate 1.Naphthol
AS-D
Myeloperoxida chloroacetate
se Granulocyte
Interpretation esterase
Cells Series 2. Alpha-
Blast ------- naphthyl
Mature cells acetate or
Myelocytic + alpha-naphthyl
MPO Interpretation 1. Specific Esterases
++ ++++ butyrate;mono
2. Non -specific Esterases
Monocytic + cytic cells are
3. Combined Esterase
++ +++ strongly
Lymphocytic = positive and is
== inhibited with
Erythrocytic = NaF;myelocyti
== c cells may be
weakly

Stains lipids positive and

such as remain weakly

sterols, neutral pos with NaF.

Sudan Black B (SBB) fats, and

Principle phospholipids. Periodic acid
These lipids oxydizes
Periodic Acid Schiff Principle
are found glycogen,
primaryly in mucoproteins
 and other high stimulated, Inc or Dec or LAP? granulocytes
molecular (CML) which
weight causes
carbohydrates decrease in
to aldehydes LAP.
to turns "bright Other causes
Pink". Pos. rxn of dec LAP:
means PNH;
lymphocytic Sideroblastic
and anemia;
erythrocytic Myeloproliferat
cells. Neg. rxn ive disorder.
in benign cells.

Acid
1.acute and phosphatase
PAS Interpretation
chronic is present in
1.Useful in diagnosing both ___ and
leukemias all
___.
2. chunky or nonerythroid
2.Look for a ____ appearance
block cells and
3.Some lymphoblast of ALL may be
3.PAS hydrolyzes
_____negative.
negative. substrate
naphthol AS-

Found in BI phosphoric

2ndary acid. The

granules of Tartrate Resistant Acid hydrolized

neutrophils Phosphatase Principle substrate

Substrate couples with a

naphthol AS- dye to form a

BI phophate is precipitate red.

Leukocyte Alkaline Phospatase hydrolyzed by All normal

LAP Principle LAP. The isoenzymes of

hydrolyzed acid

substrate phospatase

combines with are inactivated

a dye and by tartric acid

precipitates and will not

(blue and stain

black).
TRAP Interpretation 1. Hairy cell
LAP interpretation 1.a shift to the 1.In ______ leukemia, isoenzyme # leukemia

1.Used to investigate: left. 5 is resistant to tartric acid and will 2. Hairy cell

2.Neutrophils in leukomoid rxn are Malignant stain leukemia

2.Useful for confirming ____ 3. Relative
Erythrocytosis

1.polymerizati =Decreasd

on of Plasma

TDT Terminal deoxyribonucleotidal deoxynucleoti volume

Transferase: des found only

1.This is a specific cell marker that in lymphocytic 1.preleukemia
catalyzes the __________. precursors. Myelodisplastic Syndromes s
2.Not a cytochemical stain, its a 2. 1.Previously called: 2.clonal of
________ immunofluores 2._________abnormality pluripotent
3.positive in________ cent stain 3.specific immunological marker for stem cells
3.lymphocytic MDS? 3. not been
leukocytes. found

1.CML 1.Dyserythrop
2.Polycythemi oeisis
a Vera Myelodysplastic Syndromes 2.Dysmehlopo
3.Essiential Diagnostic Morphology: resis
(primary) 3.Dysmegakar
Thrombosis ocytes:
4. Agnogentic
myeloid Refractory
I.Myeloproliferative Disorders metaplasia. Anemia:
include: II. -Acute: 1.WBC <3,900
II. Classifications: acute 2. iron stores
lymphocytic increased
leukemia 3.no ringed
FAB Classifications of
-subacute: sideroblasts
Myleodysplastic Syndromes
myelodysplasti 4.rare blast in
1.RA
c syndromes blood
-chronic: 5. Neutropenia
myeloproliferat and
ive disorders thrombocytop
enia may be
1. present.
Polycythemia
vera=Malignan 1.Similar to
t increase in RA; >15%
Different Polycythemias are:
red cell mass RARS: Refractory Anemia with ringed
2.Secondary Ringed Sideroblast sideroblast in
Polycythemia= BM; iron not
hypoxia incorporated
 properly

rbc, wbc and

RAEB: Refractory Anemia with plt
Excess blast abnormalities
appear;

20 to 30%
myeloblast in
RAEB-T:Refractory Anemia with
BM; >5%
Excess blast in Transformation
myeloblast in
blood.