Special Staining Cytochemistry: BY Zenaida Capistrano-Cajucom, RMT, Ma - Ed.Mgt, Fpamet
Special Staining Cytochemistry: BY Zenaida Capistrano-Cajucom, RMT, Ma - Ed.Mgt, Fpamet
Special Staining Cytochemistry: BY Zenaida Capistrano-Cajucom, RMT, Ma - Ed.Mgt, Fpamet
CYTOCHEMISTRY
BY
ZENAIDA CAPISTRANO-CAJUCOM,
RMT, MA.ED.MGT, FPAMET
Supravital Staining
2. Evenly distributed
2. Drying
3. Heating
4. Acid pH
Physical Factors/Properties
5. Alkali pH
6. Strong fixing agents
Properties of Reticulocytes
1. More adhesive
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
counted
Retics in Square A and RBCS in
Square B
Another Miller Disk
CORRECTION OF RETIC COUNT
5. Aplastic Anemia
VARIATIONS IN NUMBER
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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).
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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).
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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.
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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
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
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+ Peroxidase: All Blasts are (+)
Reagents
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
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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Left- Positive LAP- Right- (-) LAP
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Positive LAP Test
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SUDAN BLACK REACTION
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
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Sudan Black (+)
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Fat Staining with Sudan III by Romeis
Reagents:
1. Sudan III Hollburn
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.
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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 (+)
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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
2. lymphocytes
Cell Species that are PAS + under
pathologic conditions.
1. Plasma cells
4. Pathogenic fungi-Blastomyces
brasiliensis and Histoplasma
capsulatum
5. Contaminant- starch grains
HEINZ BODY STAIN
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
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Stained Structures
1. Pappenheimer bodies
2. Siderocytes
3. Heinz Bodies
Total Reticulocytes= R + RS
END OF PRESENTATION