CYTOCHEMISTRY

 A microscopic study & identification of the chemical constituents within an individual cell

 Usefulness/Use of the Test

1. Identification of the malignant cell type on the basis of cytoplasmic & nuclear chemistry
2. Identification of cellular constituents that are present in abnormal form or abnormal amount
3. Identification of cells that lack cellular constituents
4. Identification of cells that exhibit functional abnormalities
5. Rare variants of leukemia may be recognized with the aid of cytochemical stain

 Fixatives Common to several procedures (particularly for enzymatic technique)

1. Buffered formalin-acetone (pH 6.6)

 Same buffer used in Wright’s stain: Anhydrous KH2PO4 monobasic & anhydrous Na2HPO4 dibasic are dissolved in 30 ml
distilled H2O. Add 45 ml acetone & 35 ml concentrated formalin.

2. Methanol-acetone (pH 5.4)

Groups of Cytochemical Stain

I. Enzymatic Techniques

A. Peroxidases
 An enzyme that catalyzes the oxidation of substances by hydrogen peroxide (H 2O2) A H2 + H2O2  A + 2H2O
 Used as a marker for neutrophilic granules A = represents the oxidized substance or
indicator

Diaminobenzidine (DAB) method Cyanide-Resistant Peroxidase Stain

Fixative Buffered formalin-acetone
(store at refrigerator temp.; stable for only 1 month)
Incubation (Prepare fresh for each batch) In addition to DAB method:
mixture Phophate buffer 0.07M, pH 7.4 - 50 ml Add 4.9 mg NaCN (sodium cyanide)
3,3 DAB tetrahydrochloride - 37.5 mg Titrate to ph 7.4 w/ 1 N HCl
3% H2O2

Incubate for 15 minutes Incubate for 5 minutes

Counterstain Giemsa stain, 10.0 ml
Controls Blood film from normal donor Normal blood buffy coat
(+) control = neutrophils Eosinophils – should be strongly (+) [brown]
(-) control = lymphocytes Neutrophils – (-)
Interpretation  Dark brown granules in cytoplasm of granulocytes &
monocytes
Monocyte: weak to moderate
Granulocytes: packed w/ (+) granules
RBCs: stain diffusely brown bcoz of pseudoperoxidase
activity in Hb
Etc…  This stain is used to demonstrate Auer rod in Uses: As proposed by Gabbas & Li
myeloblast & monoblast. 1. Identification of the eosinophilic component of
acute myeloid & acute myelomonocytic leukemias
 Disadvantage: it’s carcinogenic (performed w/ caution 2. Aid in recognition of de novo acute eosinophilic
using a fume hood, mask & gloves) leukemia
 B. Esterases
 Enzymes that hydrolyze aliphatic & aromatic esters at acid or neutral pH
 9 esterase bands can be demonstrated by cell electrophoresis.

Naphthol AS-D Alpha-Naphthyl Acetate Alpha-Naphthyl Butyrate Combination of Alpha-

Chloroacetate Esterase Esterase (A N A E) Esterase (B E) naphthyl butyrate -
(CE) chloroacetate esterase
Incubation  0.07 M Phosphate  0.07 M Phosphate  0.07 M phosphate  BE incubation mixture
mixture buffer, pH 7.73 (38.0 ml) buffer, pH 6.64 buffer, pH 6.69 – 38.0 ml at room temp. for 30
 Hexazotized new fuchsin  Hexazotized  Fresh hexazotized min.
(0.2 ml): Mix equal pararosanilin: Mix equal pararosanilin – 0.4 ml  CE incubation mixture
volume of new fuchsin & volume of the first 2  Alpha-naphthyl butyrate at room temp. for 5
4% Na nitrite for 1 min. sol’n for 1 min. before sol’n – 2.0 ml min.
before use. use.
 Naphthol AS-D  Alpha-naphthyl acetate
chloroacetate sol’n (2.0 sol’n: 100 mg dissolve in
ml): 10 mg Naphthol AS- 5.0 ml ethylene glycol
D chloroacetate dissolve monomethyl ether.
in 5 ml of N,N-dimethyl Refrigerate before use.
formamide. Store at 4 - *Incubate at room temp.
10C. Stable for 1 month. for 45 min.

*Incubate at room temp.

for 10 min.
Counterstain Mayer’s hematoxylin -
10 min.
Controls Normal neutrophils – (+)
control
Interpretation Bright red granules in the
cytoplasm of mast cells,
neutrophils & neutrophilic
precursor
Etc… Uses:
1. Useful in
demonstrating
myeloid elements on
paraffin-embedded
sections such as
granulocytic sarcoma
2. Identify systemic mast
cell disease
 This stain is used as a
marker for mature &
immature neutrophils, &
mast cells.
(+) controls: Normal
monocytes or histiocytes
in bone marrow or blood
film prep.
Monocytes: stain red-
brown
Lymphocytes: dotlike
 T-helper lymphocytes:
focal dotlike staining w/
long incubation
 T-lymphoblasts of acute
leukemia: focal staining
is weak or (-)
Same w/ ANAE  Normal monocytes &
neutrophils in blood
film
 Histiocytes or
developing neutrophils
in bone marrow
Enzyme activity is noted as  Cytoplasm of
dark red precipitates in the monocytes &
cytoplasm of monocytes & histiocytes – dark red
histiocytes. ppt
 Neutrophils – blue
granules
Used to differentiate acute
myelomonocytic, acute
monocytic & chronic
myelomonocytic leukemias
from other acute
nonlymphocytic leukemias
& dysmyelopoietic
syndromes.

Comparison of Esterase Substrates & Correlation with Isoenzymes

&Cell Types Isoenzymes of Acid Phosphatase Isoenzyme Positive Cells
Substrate Isoenzyme Positive Cells 0 Gaucher cells
Naphthyl AS-D 1, 2, 7, 8, 9 Mast cells, 1&4 Neutrophils & monocytes
chloroacetate neutrophils 3a Lymphocytes & platelets
esterase 3b Primitive cells & blasts
Alpha-naphthyl 3, 4, 5, 6 Monocytes, 5 Hairy cells
acetate esterase megakaryocytes
(strong), plasma cells,
lymphocytes (focal)
Alpha-naphthyl 2, 4 Monocytes,
butyrate esterase megakaryocytes
(weak), lymphocytes
(focal)
 C. Phosphatases

Acid phosphatase Tartrate-Resistant Acid Leukocyte Alkaline Phosphatase

Phosphatase (TRAP) (LAP) or Neutrophilic Alkaline
Phosphatase (NAP)
Fixative Methanol-acetate mixture - 30 Formalin 40.0 ml
seconds Methanol 360 ml
*store in freezer
Substrate sol’n Naphthol AS-BI phosphoric acid - Acetate buffer Dissolve 0.6 g of naphthol AS-BI
100.0 mg - 100 ml phosphate in 10 ml N,N-dimethyl
N,N-dimethyl formamide - Naphthol AS-BI phosphoric acid formamide. Add 0.2 M Tris buffer to
10 mg - 10 mg 2 L. Store in refrigerator.
*stable for 2 months at 4-10C N,N-dimethyl formamide
- 0.5 ml
*stable for 2 months at 4-10C
Incubation Acetate buffer - 50 ml 1.0 mg of fast garnet GBC dissolve
mixture Substrate sol’n - 0.5 ml in 10.0 ml of substrate sol’n &
Fast garnet GBC salt - 5 mg 75.0 mg of L-(+)-tartaric acid
(causes it to be tartrate-resistant)
*incubate for 45 min. . Adjust to pH 5.2. Filter before
use & use immediately. *incubate
for 1 hr.
Counterstain Mayer’s hematoxylin - 5-20 Nuclear fast red - 20 min.
min.
Mounting Glycerin jelly (water soluble)
medium
Control Peripheral blood film Normal blood film Slides taken from pregnant women in
their last trimester
Interpretation  Discrete purplish to dark red  Neoplastic cells of hairy cell  Brown granules in cytoplasm of
granules leukemia – strongly (+) neutrophils
 T-cell acute lymphoblastic  Histiocytes – may have weak Count 50 segmented neutrophils
leukemia – moderate activity tartrate-resistant acid Score staining rxn from 0-4+
confined to Golgi area phosphatase activity 0 = no granules
 Non T-cell acute leukemia – show 1+ = very few granules
(-) 2+ = moderate # of granules
scattered throughout the cell
3+ = numerous granules strating
to coalesce
4+ = cytoplasm packed w/
granules
Etc… Acid phosphatase:  Useful for leukopenic patients 0.2 M Tris buffer, pH 9.1:
 Enzyme capable of hydrolyzing  Associated w/ isoenzyme 5 Trizma base 48.44 mg
monophosphate ester at an acid Distilled H2O 200 ml
pH Adjust pH to 9.1 w/ 1 N HCl
 Present in all hematopoietic cells
& located in the lysossome Working sol’n:
Alkaline phosphatase: 50 mg of fast blue BBN dissolve in
 Group of isoenzyme that are able 50 ml of substrate, prepared fresh
to hydrolyze phosphatase ester & filter before use
at an alkaline pH
Procedure:
Cold fixatives (4-10C) – 30
seconds
Working sol’n – 20 min.
Nuclear fast red (counterstain) – 20
min.
II. Non-enzymatic technique

PAS - Schiff reagent
Colorless sol’n capable of
reacting w/ aldehyde groups in
glycogen, mucoproteins &
other high molecular weight
carbohydrates
Glycogen is predominant in
leukocytes.
(+) color – bright fuschia-pink
Reagents:
Fixative:
Formalin 10
ml
Absolute methanol 90
ml
*keep capped &
refrigerated
1% Periodic (for oxidation) –
kept refrigerated, should be
made fresh every 7 days
Schiffs reagent –
commercially prepared.
Keep refrigerated.
(Colorless; if it turns pink it
must be discarded.)
Harris hematoxylin
(counterstain)
Ammonia water – 3-5 drops
conc. NH4OH in 50 ml
distilled H2O
Sudan Black B (SBB)
For the demonstration of
certain phospholipids &
lipoproteins
Mechanism of action is
uncertain in that it may be:
a. Selective adsorption
b. A chemical rxn
c. Or a combination of both
SBB rxn is similar to the
myeloperoxidase rxn.
Advantage of SBB over the
myeloperoxidase stain:
a. Its stability to heat &
storage
b. More sensitive to
primitive myeloid cells
c. Rgts are not carcinogenic.
Disadvantages:
1. Require 1-2 hrs staining
time
2. Its specificity
3. Increased background
staining on bone marrow
2ndary to the fatty nature
of bone marrow itself
Toluidine Blue
A dye that can bind w/ acid
mucopolysaccharides in blood
cells to form metachromatic
complexes
Ex. Granules of both
basophils & mast cells are
strongly metachromatic stain
reddish violet
Usefulness: Recognition of
mast cell diseases & acute or
chronic basophilic leukemias.
?Fixative: Mota
(metachromatic complex –
reddish)
Ferric Iron - Prussian Blue
Reaction
Presence of iron granules in
otherwise mature,
nonnucleated red cells
(siderocytes) may be seen in
iron overload or poor iron
utilization syndromes
Reagents:
Fixative: absolute methyl
alcohol
Interpretation:
Bone marrow iron storage is
usually reported as absent,
decreased, increased or
normal.
Other principal use of the iron
stain in hematology is the
evaluation of red cell iron
utilization.
Troubleshooting:
Presence of excessive
amounts of extracellular
material that stains (+) for
iron can be caused by
contaminated rgts.
(-) staining in a (+) control
may be 2ndary to improper
pH of the K ferrocyanide

III. Immuno Cytochemical Techniques

 There is an antigen-antibody rxn. Advantages:

 Immunochemistry – the identification of the
immunologic phenotype of a given cell population through
the use of specific monoclonal or polyclonal antibodies
against selected cell antigens
 Specimens used: Cell suspension, Paraffin or cryostat
section, Smears, Imprints, Cytospin prep
 A slide-based technique is advantageous bcoz it allows
for counting of designated cell populations & permanent
slide can be filed for future review.
 Both practical & cost effective
 No expensive equipment is required.
 The specimen may be as much as 1 week old.
 Application of this technique in hematology lab has
been primarily for the identification of the cell types
involved in acute or chronic leukemias.
 Enzyme Immunocytochemistry (Immunoperoxidase)
 Peroxidase – antiperoxidase (PAP)
 Plant enzyme used – horseradish peroxidase
1. It is easily obtainable & stable
2. Endogenous peroxidase is easily blocked
3. A variety of chromogens are available to react w/
peroxidase to form a colored end product
4. Inexpensive
 Immunoalkaline phosphatase – another enzyme
Advantage: lack of cross reactivity w/ the pseudoperoxidase
of erythrocytes or granulocytes
 PAP technique uses 3 rgts:
1. Primary antibody that is specific for the antigen in question
2. 2ndary Ab that will bind to the Fe portion of the 1 Ab &
carries the PAP complex as a tag
3. Substrate used to make the peroxidase rxn visible
 Method – depend on the kit commercially available
 Control – buffy coat preparation made from peripheral
blood from healthy individual