SPECIAL STAINING

CYTOCHEMISTRY

BY
ZENAIDA CAPISTRANO-CAJUCOM,
RMT, MA.ED.MGT, FPAMET
 Supravital Staining

It demonstrates cells in the living

condition.
It demonstrates the coarse skein or
the network of filaments.
It represents the precipitated RNA.
It Wright’s stain, it is called
Polychromatophilic Cell.
Appearance of Reticulocyte
1. Narrow band traversing the cell

2. Evenly distributed

3. Densely packed like a nucleus

Physical Factors Affecting the

Reticulum
1. Concentration of the dye

2. Drying

3. Heating

4. Acid pH
 Physical Factors/Properties

5. Alkali pH
6. Strong fixing agents

7. Glucose and Sodium inhibit reticulum

Properties of Reticulocytes
1. More adhesive

2. Specific Gravity is low

Reticulocyte in NMBN
Retics
Retics
9
 In a Wright's-stained
blood smear of a
regenerative anemia in a
cat (top image), the larger
purple cell (P) is a
polychromatophil which
corresponds to an
aggregate reticulocyte (A)
in the new methylene
blue-stained smear
(bottom image). In
contrast, the larger red
cell is a macrocyte (M)
that corresponds to a
punctate reticulocyte
(PR).
10
 Methods
A. New Methylene Blue N
B. Seiverd’ Method
C. Cook, Meyer and Tureen
D. Rapid Method of Schilling
E. Sabin’s Method
F. Wolfer’s Method
Results: Substancia
granulofilamentosa= blue
RBC- Green
Mixing of Blood And Staining Solution

1. Capillary Method
2. Test Tube Method
3. Slide Method
-Standing Time: 10 minutes
-NMBN is superior than BCB
-Potassium Oxalate –isotonic
- equal amount of blood & stain
 COUNTING OF CELLS
Terms Used: Reticulocytosis
Reticulocytopenia
Reference Values: 0.5-1.5% Adults
0.5- 2% Children
4.5-6.5% Infants

Retic (%) = R X 100

1000
Miller Disk How cells are

counted
 Retics in Square A and RBCS in
Square B
Another Miller Disk
CORRECTION OF RETIC COUNT

CRC = U.R. X Patient’s Hct

Average Hct

RPI = Corrected Retic Count

Maturation Time
Reticulocyte Production Index
Hct Maturation Time
0.45 +- 0.05 1.0
0.35 +- 0.05 1.5
0.25 +- 0.05 2.0
0.15 +- 0.05 2.5

RPI = Corrected R= 4/2 MT= 2

RBC production is Increased
 INDICATIONS

1. Lead Poisoning & Hemolytic

Icterus
2. Response to PA to Vit B12
3. Acute Hemorrhage

4. Hemolytic Anemia- HDN

5. Aplastic Anemia
 VARIATIONS IN NUMBER

1. Decreased in: BM depression

2. Increased in: hemorrhage
- splenectomy
- hemolytic anemais
- after specific treatment in anemia
 Automation ( Hematology
Analyzer)
 Reticulocytes are counted by
staining blood a fluorescent dye
(Oxazine 750). The immature red
blood cells are then detected by
the degree of fluorescence
(mature erythrocytes will not
fluoresce).

22
23
Right: Red Cell Volume by Hgb
Scatterplot
 The Y-axis represents red cell
volume and the X-axis represents
red cell hemoglobin. Normocytic
normochromic red blood cells fall in
the center box (green
arrowhead), normal red blood cells
into the upper left hand box (larger
volume, lower hemoglobin, red
arrow).

24
 whereas polychromatophilic
erythrocytes (which are larger and
have less hemoglobin than mature
red blood cells) stream off the
cluster of normal red blood cells
into the upper left hand box (larger
volume, lower hemoglobin, red
arrow).

25
 Left: Reticulocyte scatter plot.
This represents the degree of
oxazine staining (RNA) in the
erythrocytes. Mature red cells
(mature) take up very little
oxazine. Reticulocytes have varying
degrees of fluorescence, resulting in
low (A), intermediate (B) and
high (C) levels of fluorescence.

26
 PEROXIDASE REACTION

Principle: Demonstration of
peroxidase granules depends on
their content of an iron porphyrin
compound containing an enzyme
peroxidase which promotes the
oxidation of benzidine by
hydrogen peroxide.
 Methods

1. Goodpasture Method
2. Osgood and Asthworth Method
3. Sato and Sekiya Method
4. Kaplow’s Myeloperoxidase Method

Fixative used: Formaldehyde

Absolute Ethyl Alcohol
Reagents

Zinc Sulfate solution

Incubation Mixture
Ethyl Alcohol
Benzidine Hydrochloride
Zinc Sulfate
Sodium Acetate
Procedure

1. Blood Smear
2. Fix: 60 seconds in 10% formol
alcohol
3. Incubation mixture= 30 seconds
at room temperature
4. Wash for 5 to l0 seconds
5. Air dry and mount
Comment: To preserve the activity, it is
kept in the dark for several weeks. It is
called Graham Knoll Reaction.
Interpretations:
1. Peroxidase Positive Granules are seen in:
Promyelocytes, Myelocytes,
Metamyelocytes, Neutrophils, Eosinophils
and monocytes
2. Peroxidase negative granules
myeloblasts, lymphoblasts,
plasma cells and basophils
3. Granules in Promyelocytes
(blue green)
4. Monocytes= less intensely
5. PMN- abundant and blue black
6. Non-hemopoietic cells of the body like
tumor cells are consistently peroxidase
negative.
Peroxidase Reaction Interpretations
Increased:
1. Neutro- greatest, Eosi-moderate

2. Baso in leukemia (+)

3. Mono- finely granular

4. Auer rods- myeloblasts and mono

5. Increased in CML but decreased in

AML, Decreased also in PMN of
infections
Decreased ( No Activity)
1. lymphocytes
2. RBCs
3. Mature Basophils
4. Acute Lymphocytic leukemia
Auer Rods in Myeloblasts

35
+ Peroxidase: All Blasts are (+)
Reagents

1. p- phenylene diamine and catechol

reagents
2. O- toluidine
3. 2-6 dichlorophenol indophenol
4. Note: Benzidine is not used now
because of its carcinogenic
effect.
 LEUKOCYTE ALKALINE
PHOSPHATASE
Principle: The reaction depends on
the hydrolysis of alpha-napthyl
phosphate by alkaline
phosphatase to produce a
colored precipitate with
diazotized amine.
Procedure:
1. Thin smear is prepared.
2. Fix for 15 secs at Rm temp in
buffered acetone
3. Wash gently w/ running H2O for 30
to 60 seconds and air dry.
4. Cover with incubation mixture for
10 minutes.
5. Wash gently for 30 to 60 seconds
6. Counterstain with Mayer’s
Hematoxylin
7. Wash again
8. Mount the cover glass on a slide
with a small drop of immersion oil.
Control Slides
1. High Values. . . Pregnant Women
2. Low Values . . . Patient’s with
Untreated Myeloid Leukemia
 Composition of Substrate Solution
-Sodium Alpha-Napthyl Phosphate
-Brestamine Fast Garnet Salt
-Working buffer
- 2 amino 2 methyl propane diol in
distilled water.
Mayer’s Hematoxylin
- Hematoxylin Sodium Iodate
- Distilled Water Aluminum
Potassium Sulfate
Interpretations

1. + color= pink, ruby red, reddish

brown granules
2. Cytoplasm- red, Nucleus- black
3. Alkaline Phosphatase activity-
found in PMN and Metamyelocytes
Alkaline Phosphatase Activity
1. Neutrophils 3. Small degree “B”

2. Band lymphocytes
Increased in Leukemoid Reactions
Decreased in C M L
Increased in Leukemoid Reactions:
1. Pregnancy -myeloschlerosis
2. Polycythemia vera
3. Multiple myeloma
4. People using cortocisteroids
5. Hodgkin’s disease
6. Aplastic Anemia

7. Myelofibrosis -malignant growth

 Decreased in:
1. C M L
2. Hereditary Hypophastasis
3. P N H
4. Sickle Cell Anemia
5. Marked Eosinophilia
6. Siderablastic Anemia
Normal Activity in:
1. Untreated HA 3. Viral Hepatitis
2. Lymphosarcoma 4. Secondary
Polycythemia
Kaplow’s Scoring

Kaplow’s Score= No. of + 1 cells X 1

+ No of (+ 2) cells X 2 + No of (+
3 cells) X 3 X No of (+4 cells) X 4.

Normal= 15 to l00
Less than 15- Leukemia
Increased in L.R.
Positive LAP Test
Neutrophil with a score of 4 and a
Neutrophil with a score of 1
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48
49
50
Left- Positive LAP- Right- (-) LAP

51
Positive LAP Test

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 SUDAN BLACK REACTION

This demonstrates lipid granules in

leukocytes. Lipid granules appear
dense black.
Lipids
1. Sterols

2. Phospholipids

3. Neutral Fats
- More sensitive than the Chloroacetate
esterase staining of myeloblasts
- Vacuolated immature cells in Burkitt’s
Lymphoma may show + staining of lipids
present in the vacuoles.
Procedure:
1. Fix air dried films-formalin vapor-10min.
2. Treat with the working solution for 30 mins
at 37 deg.C.
3. Wash with abs.alcohol and distilled H2O

4. Counterstain with Giemsa stain for 1 hr.

5. Mount with cover glass and examine directly.

Sudan Black B Positive

56
Sudan Black (+)

57
Fat Staining with Sudan III by Romeis

Reagents:
1. Sudan III Hollburn

2. Mayer’s or Erhlich”s Haemalum

Procedure:
1. Fix for 25 minutes with 1:9
formalin solution, Rinse with tap
H2O, immerse in distilled H2O 3 X
and drip off.
2. Immerse in Sudan Solution at
Rm temp for 6 to 8 hours.
3. Wash thoroughly with distilled
H2O.
4. Treat with Haemalum for 15 to 20
min.
5. Wash for half an hour.

6. Drip off and moist with glycerin

jelly under a cover slip.
Results:
1. Eosinophils- strongly positive
2. Neutrophils- positive (orange)
3. Monocytes are + only in part or in
varying degrees
4. Negative more or less: Baso,
lympho, megakaryocytes, platelets,
plasma cells and erythrocytes
5. After meals: droplets maybe seen
6. Vacuoles of Plasma cells yield no
fat staining since they are enclosed
with proteinic substance (Russell
Bodies)
+ Sudan Black in Blasts
+ Sudan Black
 FEULGEN REACTION
This demonstrates DNA. Nucleoli will
not stain but bodies of nuclear
origin will.
1. Fix in 9 parts methanol and 1 part
of formalin.
2. Rinse in distilled water.
3. Place in normal Hcl for 15 min at
56 deg.C. If smears tend to
detach from the slide, 0.1N Hcl
maybe used.
4. Stain in 1 to 10 Wright’s stain fro
30 to 60 minutes.
Feulgen Reaction

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 INCLUSION BODY STAIN
This demonstrates Heinz Like
Inclusion bodies seen in
association with
Hemoglobinopathies, Hb H and
Hb Zurich.
Procedure:
1. 1 volume + 1 vol of BCB
2. Incubated at 37 deg.C for 15 mins.
3. Place a small drop on the slide and
examine.
 Differences: Hgb H inclusion and Hgb
Zurich
1. Hgb H inclusion=denatured Hgb
H
= multiple, small, irregularly
shaped blue dots.
= not seen in Wright’s stained
smears
2. Hgb Zurich= single, unusually
large blue bodies,
= visible in Wright’s stained
blood smears
PERIODIC ACID SCHIFF STAIN
Principle: Aldehyde groupings arise
when glycoside oxidized with
periodic acid and stains as basic
fuchsin reacts and yields a
characteristic color(red) reactions
in the presence of aldehydes.
Procedure:
1. Prepare a thin smear of the bone
marrow in the usual manner.
2. Fix in formalin-ethanol for 15
minutes.
3. Wash briefly with water.
4. Cover with periodic acid solution for
10 minutes.
5. Wash briefly with tap water and air
dry.
6. Stain with Schiff’s Basic Fuchsin for
30 minutes.
7. Cover with Sulfate water for 5
minut6es. Drain off the Sulfate and
repeat the procedure 4 times.
8. Wash in tap water for 5 minutes.
9. Counterstain with aqueous
hematoxylin for 15 minutes.
10. Wash in tap water fro 5 minutes and
air dry.
11. Mount the cover glass on the slide
with a small drop of immersion oil.
CHO that give + results
1. Monosacharides 4. inositol

2. Polysacharaides 5. mucoprotein

3. Glycoprotein conjugates
1. Used to diagnose Erythroileukemia
or the D Guglielmo’s Disease.
2. This will differentiate Erythremic
myelosis form siderablastic
anemia.
3. Cells are counted based on 1+, 2+,
3+
4. Only Erythroid cells are counted
Conditions
1. Normal ( Negative)
2. Pernicious Anemia ( Negative)
3. Hemolytic Anemia ( - or Slight +)
4. Sideroachrestic Anemia( - or Sl.+)
5. Iron Deficiency ( Intense)
6. Thalassemia major (Intense)
7. Erythroleukemia (intense)
PAS Reaction by Hotchkiss and Mc
Manus, Modified by Merker
Procedure:
1. Fix for 5 minutes in 14% formalin-
alcohol.
2. Immerse in periodic acid solution
for 10 minutes
3. Immerse in rinsing fluid for 5
minutes
4. Immerse in Schiff’s reagent at
room temperature in a dark place
for 15 minutes.
Positive PAS
PAS ( Periodic Schiff (+)

74
5. Three passages of 2 min.each
through rinsing fluid Rinse for 10
min with running water tap water,
then air dry.
6. Immerse in Harris Haematoxylin for
2 to 3 minutes. Rinse with tap
water and air dry.
Under normal conditions, there is
an increase of polysaccharides as
the cells mature.
They occur in large numbers in
Haemoblastoses
Cells that contain Polysaccharides
without exception
1. Blood Basophils

2. Neutrophils

3. Megakaryocytes

4. Platelets

Cells that contain a certain % of

Polysaccharide
1. Monocytes

2. lymphocytes
Cell Species that are PAS + under
pathologic conditions.
1. Plasma cells

2. Erythrocytes from proerythroblasts

to non-nucleated erythrocytes
3. Carcinoma cells

4. Pathogenic fungi-Blastomyces
brasiliensis and Histoplasma
capsulatum
5. Contaminant- starch grains
 HEINZ BODY STAIN

Glutathione Instability Test by

Beutler
1. Incubate a mixture of blood and

acetylphenylhydrazine at 37
deg. C. for 15 minutes.
2. Transfer a drop of the blood from
the (1) and add a drop of brilliant
cresyl blue or 2% crystal violet.
Interpretations
1. Heinz bodies appear a deep
purple, irregularly shaped bodies
varying in size from dots to
spheres, 2u in diameter. One or
more maybe present in the cell.
2. They are eccentrically placed
and lie close to the cell membrane.
3. They may protrude from the
erythrocyte by a stalk or occur free
in the plasma.
They are prominent in:
1. Splenectomized individuals

2. Congenital Heinz Body Anemia

3. Thalassemia major (Normoblasts,

reticulocytes and mature
erythrocytes)
Heinz Bodies H.B. in G6PD
 PRUSSIAN BLUE REACTION
Iron stain is useful for differentiation
of anemia due to iron deficiency
anemia and anemia of
Thalassemia or other disorders in
which iron accumulates because it
is poorly utilized for hemoglobin
synthesis.
Reagents:
20% Potassium ferrocyanide
Conc. Hcl
Prussian Blue Reagent: Conc. Hcl
is added to an aliquot of Potassium
ferrocyanide reagent until a white
precipitate forms.
Douglas and Dacie’s Method
1. Prepare thin smears in the usual
manner
2. Fix the smear in absolute alcohol
methyl alcohol for l0 minutes.
3. Rinse with demineralized water
and air dry.
4. Stain with Prussian Blue
Reagent for 30 minutes.
 Siderocytes -->RBC's containing
Iron Granules -->Seen in patients
with iron overload in body (Ex.
Thalassemia, Hematocrosis)
Seen in Patients with Iron
Overload Syndrome
Howell Jolly Bodies
Howell Jolly Bodies & Heinz Bodies
5. Wash for at least 4 minutes in a gentle
stream of demineralized water and air
dry.
6. Counterstain with dilute Safranin O for
1-5 minutes.
7. Rinse

8. Mount a cover glass on the slide with a

small drop of immersion oil.
Calculation:
% of Siderocytes= No. of S X 100

1000
 Stained Structures
1. Pappenheimer bodies

2. Siderocytes

3. Heinz Bodies

(+) Result= Vivid Blue or Blue

Green
- The granules vary in size and
number.
( within the range of visibility or
1-2 u)
- 1 to 2 in number or one dozen
A. Bone Marrow B. Erythroblast
Pappenheimer Bodies and B.
Stippling
Calculations:
Total Siderocytes = S + RS

Total Reticulocytes= R + RS
END OF PRESENTATION