Periodic Acid Schiff (PAS)

Sudan Black B (SBB)

Prussian Blue Reaction
3. IMMUNOCYTOCHEMISTRY

CYTOCHEMISTRY by Brian M. Denney, RMT

Cytochemistry the application of normal processes

to cells and tissues to reveal their chemical
composition
Useful in the ID of:
Malignant Cell types (both nuclear and
cytoplasmic constituents)
Cellular constituents (abnormal/large
amounts)
Lack of cellular constituents
Cells exhibiting functional abnormalities
Acceptable specimen and fixatives:
Well- made peripheral blood film
Bone Marrow Smears
Smears from Lymph nodes and spleen
Adequately and properly fixed well done smears is a
must
Concentration methods/ Cytocentrifuge preparations
is done when there is insufficient amount of
specimen
A good fixative must contain:
ACETONE/ALCOHOL/FORMALDEHYDE or a COMBO of
the 3
One must consider the age of the specimen
PBS must be air dried at:
Minimum = 4 hours @RT
Maximum = 48 hours @ RT
*Failure to air dry beyond or below the
specified time may cause leeching of the
stain
Cytochemistry is divided in to three groups:
1. ENZYMATIC
Phosphatases
Esterases
Peroxidases/Myeloperoxidases (MPO)
2. NONENZYMATIC

@helloktelle

Page 1

*Endpoint = color production

ENZYMATIC TECHNIQUES
PHOSPHATASES
May be Alkaline or Acid
Are groups of isoenzymes that are capable of
hydrolysing monophosphate esters at a particular pH
Substrate: -naphthyl

General Equation:
-naphthyl Phosphatases

- naphthol

-naphthol + Diazo reagent (colorless)

Azodye (colored)
*The color depends on the substrate
- present in all hematopoietic cells
- used in preliminary ID of acute T-cell lymphocytic
leukemia (ALL)
- contains seven (7) noneythroid isoenzymes

Isoezyme 0 & 4 Gaucher cells

Isoenyzme 1 Neutrophils and Monocytes
Isoenyzme 2 not mentioned
Isoenzyme 3 (3A) Lymphocytes and platelets
Isoenyzme 3B Primitive cells and blasts
Isoenyzme 5 Hairy cells
Reagents:
o Fixative of choice: cold methanol acetone &/or
citrate acetone mixture

Acetate Buffer (ph 5.2) made up of 10.75 mL

sodium acetate, 21 mL 1N acetic acid (keeps
the buffer acidic) and 1000 mL distilled H20
o Staining substrate (the one acted upon by the
enzyme) 100 g of Naphthol AS-BI phosphate
and 10 mL N,N, -dimethylformamide
o Fast garnet GBC primary stain/ coupling
reagent
o Mayers Hematoxylin secondary/
counterstain
o Incubation Mixture 50 mL of acetate buffer
+ 0.5 mL substrate + 5 mg of Fast Garnet
GBC salt
o Mounting media glycerine jelly (any other
mounting media will cause leeching out of
Fast Garnet GBC)
PROCEDURE:
1) Smear is fixed for 30 seconds
2) Placed in the incubation mixture for 45
minutes
3) Placed in Mayers hematoxylin for 15-20
seconds
4) Wash
5) Air dry
6) Mount with glycerine jelly

Lymphocytes in chronic lymphocytic leukemia

Lymphocytes in lymphosarcoma

Tartrate Resistant Acid Phosphatase (TRAP)

Used in the diagnosis of Hairy cell leukemia
L( + ) tartaric acid is added to the substrate solution
which will inhibit all other isoenzymes other than
isoenzyme 5, which is resistant, thus producing a
negative staining reaction
Interpretation:
Hematopoietic cells = NEGATIVE
Hairy Cell Leukemia = POSITIVE
Some cells may be PARTIALLY inhibited (faint
staining):
Atypical lymphocytes in infectious
mononucleosis

@helloktelle

Page 2

Leukocyte Alkaline Phosphatase (LAP) a.k.a.

Neutrophil Alkaline Phosphatase (NAP)
Used in differentiating CML from leukemoid reaction
Reagents:
o Fixative: citrate formaldehyde acetone
o Tris buffer maintained at a pH 9.1
o Staining substrate ( same as ACP)
o Fast blue BB or Fast Red violet LB primary
stain
o Neutral red (fast blue) or Gill No. Hematoxylin
(fast red violet) counterstain
o Incubation mixture (same as ACP)
* Incubation mixture is = buffer + substrate +
primary stain
Result:
Blue-black (fast blue)
Red precipitate (fast red violet)
Interpretation:
Mature neutrophils and bands = POSITIVE
(presence of LAP)
All other leukocytes = NEGATIVE
PROCEDURE:
1) Examine 100 mature neutrophils and bands
2) Grade each cell
3) Count the number of cells for each grade
4) Multiply the number of cells by their grade
5) Add all the products to obtain the LAP score
Score
0
1+

2+

LAP SCORE CHART

Description
No granules in the cytoplasm
No staining
Few small granules in the cytoplasm
(could be counted)
Faint and diffuse staining
Moderate staining granules in 50-80% of

3+

4+

Normal value (varies):

13-160, 20-100, 40-100,30-185
Reporting:
Increased score :
- polycythemia vera
- leukemoid reactions
- Infection
- 3rd trimester of pregnancy
- CML blast crisis
- CML with infection
Decreased score:
- Paroxysmal Nocturnal hemoglobinuria
- Marked eosinophilia
- Sickle cell anemia
- CML
- Sideroblastic Leukemia
ESTERASES
are a group of isoenzymes that are capable of
hydrolysing aliphatic and aromatic esters at an acid
or neutral pH
Substrate: naphthyl esters
Used to differentiate acute leukemias that are
myeloid from monocytic in origin
Consists of nine (9) isoenzymes with specific cellular
location:

@helloktelle

SPECIFIC ESTERASES- Isoenzyme 1, 2, 7, 8

and 9 = localized/ abundant in neutrophils &
mast cells
NONSPECIFIC ESTERASES - Isoenzyme 3,4,5
and 6 = localized/abundant in monocytes,
megakaryocytes (very strong reaction) ,
plasma cells (just positive) and lymphocytes
(focal)
*Focal a precipitate that forms a single-dot
like pattern that is eccentric to the cells
cytoplasm

cytoplasm
Pale, with a moderate amount of blue
staining
Strongly stained granules in 80-100% of
cytoplasm and beginning to coalesce
Strong blue precipitated staining
Strongly stained granules packed into the
cytoplasm (nucleus is the only visible
component)
Deep blue or brilliant staining with no
visible cytoplasm

Page 3

General equation:
Esterase
Naphthyl esters
naphthol
H20
Napthol + diazonium salt/ diazo dye (colorless) colored
azodye
Naphthol AS-D Chloroacetate esterase
- substrate: Naphthol AS-D Chloroacetate
- used to demonstrate specific esterases
- to distinguish myeloid leukemias from monocytic
leukemias
- very stable enzyme; can still be visualized in paraffin
embedded sections even after months (i.e. myeloid cells in
granulocytic sarcoma and mast cells in systemic mast cell
disease)
Reagents:
o
o
o
o
o

Fixative: Citrate-acetone formaldehyde

Sodium nitrite solution (4%) 200 mg of sodium
nitrite dissolved in 5 mL distilled H2O
Staining substrate substrate dissolved in N,N, dimethyl formamide (solvent)
Fast red violet primary stain
Mayers hematoxylin or Gill No. 3 counterstain

Result : red cell granulation

Interpretation:
Myeloid cells = POSITIVE
Monocytic cells = NEGATIVE (-) or WEAKLY (+)

ALPHA- NAPHTHYL ACETATE ESTERASE (ANAE)

- Used to demonstrate nonspecific esterases
- Substrate: alpha-naphthyl acetate
- to distinguish monocytic leukemia from myeloid leukemia
Reagents:
o
o
o
o
o

Myeloid and lymphoid cells = NEGATIVE

Do not stain plasma cells, lymphoblasts and
megakaryoblasts
Isoenyzme 2 & 4
- monocytes
- weak rxn with megakaryocytes
- focal reaction with lymphocytes
- no reaction with plasma cells

Fixative citrate acetone formaldehyde

Sodium nitrite (4%)
Staining substrate substrate dissolved in EGME
(ethylene glycol monomethyl ether)
Fast blue BB primary stain
Mayers hematoxylin or Gill No. 3

PEROXIDASE (MYELOPEROXIDASE)
- are enzymes that catalyse the oxidation of substrates in
the presence of hydrogen peroxide
- are present in the primary granules of neutrophils,
eosinophils and to lesser extent monocytes while absent in
lymphocytes and in all eythroid lines
- used in differentiating acute myelogenous leukemia and
acute monocytic leukemia from ALL
- best cytochemical stain for auer rods ( Faggot cell- bundle
of auer rod)

Result: brown to dark brown

Recommended substrate: 3,3- diaminobenzidine (DAB)

Interpretation:

MPO
3,3- DAB + Hydrogen peroxide (oxidizing
agent)
colored precipitate (made up of quinone rings) + H2O (by
product)

Monocytic cells = POSITIVE

Myeloid and lymphoid cells = NEGATIVE
Erythroid cells = WEAKLY (+)

ALPHA-NAPHTHYL BUTRYRATE ESTERASE (ANBE)

-

Used to demonstrate nonspecific esterase esp.

Isoenzyme 2 and 4
Substrate: alpha-naphthyl butyrate
To distinguish monocytic leukemia from myeloid
leukemia
Reagents: same with ANAE except the substrate used
Interpretation:
Monocytic cells = POSITIVE

@helloktelle

Page 4

Reagent:
o
o
o

Fixative: cold formalin/acetone or glutaraldehyde

Staining substrate 37.5 mL 3,3- DAB + 0.15 mL
H2O2
Gill No. 3 Hematoxylin

Result: Gray black granules in the cytoplasm while

eosinophils will stain red brown
Interpretation:

Myeloid cells = POSITIVE

Monocytic cells = weakly (+) or NEGATIVE
Lymphoid and Erythroid cells = NEGATIVE
NON ENZYMATIC TECHNIQUES

Periodic Acid Schiff (PAS)

-

Stains mucoproteins, glycoproteins and high

molecular weight carbohydrates (esp. glycogen)
Aids in the diagnosis of ALLs and M6
Used to confirm the presence of glycogen in the cells
Involves 2 steps:
Periodic acid is used to form aldehyde groups
from glycogen
Glycogen + periodic acid aldehydes
Aldehyde groups react with Schiff reagent
producing a bright reddish pink color
Aldehyde + colourless Schiffs rgt. insoluble bright
reddish pink ppt. /red ppt. (known as aldehydefuschin-sulfurous acid compound)
Reagents:
o
o
o

@helloktelle

Fixative : formalin/ethanol
Periodic acid (1%) 1 g periodic acid
dissolved in 100 mL distilled H2O
Schiff Reagent if becomes pink = expired
- to see if still viable- add several drops of
Schiff reagent + 10 mL of 37% formaldehyde
- Light purple = viable ; Colorless = not viable

Page 5

Sudan Black B (SBB)

-

Used to demonstrate lipids (lipoproteins &

phospholipids) present in the cells
Useful in distinguishing AML from ALL
Reagents:
o Fixative: buffered formalin/ acetone (ph 6.6)
o SBB reagent (0.3 g in 100 mL absolute
alcohol)
o Phosphate Buffer
o Harris Hematoxylin
Interpretation
Myeloid cells = POSITIVE
Monocytic cells = NEGATIVE/ WEAKLY (+)
Lymphoid cells = NEGATIVE
Prussian Blue Reaction

Staining procedure for iron stores in the bone marrow

Quantitated by using Pearls Reagent (potassium
ferricyanide)
Indicated by a blue-green precipitate
Immunocytochemistry
The ID of the immunologic phenotype of a given cell
population through the use of specific
monoclonal/polyclonal antibodies against selected
cell antigens
Factor VIII antibodies used for accurate diagnosis of
M7