Understanding Special Stains in Pathology

 
SPECIAL STAINS
 
DR HUZLINDA HUSSIN
DEPT OF PATHOLOGY
13 MARCH 2018
 
H
&
E
 
s
t
a
i
n
 
i
s
 
r
o
u
t
i
n
e
 
s
t
a
i
n
.
-
It 
is
 
the
 
preliminary
 
or
 
the
 
first
 
stain
 
applied
 
to
 
the
 
tissue
sections
-
Gives
 
diagnostic
 
information
 
in
 
most
 
cases.
 
 
A
 
s
p
e
c
i
a
l
 
s
t
a
i
n
 
i
s
 
a
 
s
t
a
i
n
i
n
g
 
t
e
c
h
n
i
q
u
e
 
t
o
 
h
i
g
h
l
i
g
h
t
v
a
r
i
o
u
s
 
i
n
d
i
v
i
d
u
a
l
 
t
i
s
s
u
e
 
c
o
m
p
o
n
e
n
t
 
o
n
c
e
 
w
e
 
h
a
v
e
p
r
e
l
i
m
i
n
a
r
y
 
i
n
f
o
r
m
a
t
i
o
n
 
f
r
o
m
 
t
h
e
 
H
&
E
 
s
t
a
i
n
 
CLASSIFICATION:
 
1.
S
t
a
i
n
s
 
f
o
r
 
c
a
r
b
o
h
y
d
r
a
t
e
s
 
2.
S
t
a
i
n
s
 
f
o
r
 
a
m
y
l
o
i
d
 
3.
N
u
c
l
e
i
c
 
a
c
i
d
 
s
t
a
i
n
s
 
4.
L
i
p
i
d
 
s
t
a
i
n
s
 
5.
S
t
a
i
n
s
 
f
o
r
 
m
i
c
r
o
o
r
g
a
n
i
s
m
s
 
6.
C
o
n
n
e
c
t
i
v
e
 
t
i
s
s
u
e
 
s
t
a
i
n
s
 
7.
S
t
a
i
n
s
 
f
o
r
 
p
i
g
m
e
n
t
s
 
a
n
d
 
 
m
i
n
e
r
a
l
s
 
CARBOHYDRATES
 
SIMPLE
 
CARBOHYDRATES
(molecules
 
composed
 
purely
 
of
carbohydrates)
 
Monosaccharides
(glucose,mannose,galactose)
 
O
l
i
g
o
s
ac
c
hari
d
es
(sucrose,maltose)
 
Polysaccharides
(glycogen,starch)
 
GLYCOCONJUGATES
(molecules 
composed 
of
carbohydrates 
and 
other
molecules 
such
 
as 
protein
and
 
lipid)
 
Proteoglycans
 
Mucins
 
Others
 
glycoproteins
 
1. STAINS FOR CARBOHYDRATE
 
1.
Periodic
 
acid
 
schiff
(PAS)
 
technique
2.
Alcian 
blue
 
method
3.
Combined 
alcian 
blue-
 
PAS 
techhnique
4.
Mucicarmine
 
technique
5.
Colloidal 
iron
 
technique
6.
Metachromatic
 
staining
7.
Iodine 
staining 
for
 
glycogen
8.
Enzymatic 
digestion
 
technique  
Diastase 
digestion
,
Sialidase 
digestion,  
Hyaluronidase
 
digestion
 
Glycogen in Ewing's sarcoma, PAS stain
 
This is nodular glomerulosclerosis involving a
glomerulus in the kidney, as seen with the PAS stain.
 
Candida in lung, PAS stain.
 
Note PAS +ve cytoplasmic granules partly resistant to
diastase
 
The goblet cells of the gastrointestinal tract are filled
with abundant acid mucin and stain pale blue with
this Alcian blue stain.
 
Alcian blue/ PAS stain
Acid mucins - blue.
Neutral mucins - magenta.
Mixtures - purple.
Nuclei - grey/blue.
 
C
R
YP
TOC
O
CCUS  
STAINED 
BY
MUCICARMINE
 
The cytoplasm of the cells lining this neoplastic gland in a colonic
adenocarcinoma are pink with the mucicarmine stain, indicative of
mucin production typical for an adenocarcinoma at this site
 
2. STAIN FOR AMYLOID
I. Congo red
-
Also called amyloid stain
-Must examine stained tissue with standard and
polarized light
-Amyloid under polarized light has apple green
birefringence, based on the molecule being in an
antiparallel beta-pleated sheet.
- amyloid-red; nuclei- blue
AMYLOID
 
Extracellular
 
,
 
amorphous
 
,
 
eosinophilic
material
 
Composed
 
of
 
protein
 
in
 
an
 
antiparallel
 
 
-
pleated 
sheet
 
configuration
 
In
 
H&E
 
stain
 
,
 
can
 
be
 
confused
 
with
 
hyaline
and 
fibrinoid
 
substances
 
Earliest
 
special
 
stain
 
used
 
for
 
amyloid
 
was
Iodine by
 
Virchow
 
Amyloid displays apple-green birefringence under
polarized light.
 
AMYLOID 
BY 
CONGO
 
RED STAIN UNDER
L
IGHT MICROSCOPE
 
3. Stains for lipid
 
I.
Oil Red O
II.
Sudan black B
 
LIPIDS
 
SIMPLE
 
LIPIDS
 
-
FATS
-
OILS
-
W
A
XES
 
COMP
O
UND
LIPIDS
 
- 
c/o 
fatty 
acids,
alcohol 
and 
one
more
 
group
 
such
 
as
phosphorus 
or
nitrogen
 
DERIVED
 
LIPIDS
 
-
Derived 
from
simple 
or
compound
 
lipids
by
 
hydrolysis
-
cholesterol
-
Bile
 
acids
 
 
Simple
 
lipid
 
is
 
best
 
demonstrated
 
with
 
fresh
 
frozen
sections
 
Best
 
fixative
 
 
Formal
 
calcium
 
(2%
 
calcium
 
acetate
 
+
10%
 
formalin)
 
I. Oil Red O
 
Stain identifies neutral lipids and fatty acids
Fresh smears / cryostat sections of tissue are necessary because alcohols used in tissue
processing remove lipids
Rapid and simple routine stain
Uses:
Differentiate fibroma from thecoma (not that important a distinction)
Diagnose renal cell carcinoma, sebaceous gland tumors of skin, lipid-rich carcinomas
Identify fat emboli in lung tissue or clot sections of peripheral blood
 
Oil red O stain of fat emboli in lung.
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Special stains in pathology provide crucial diagnostic information beyond routine stains like H&E. They help highlight specific tissue components like carbohydrates, amyloid, nucleic acids, lipids, microorganisms, connective tissues, pigments, and minerals. This article delves into the classification of special stains and focuses on carbohydrates, explaining the types of carbohydrates and techniques for carbohydrate staining. The Periodic acid Schiff (PAS) technique, Alcian blue method, Mucicarmine technique, and other staining methods are discussed in detail, along with the components stained and their possible uses in pathology. Various examples of tissues and structures stained with carbohydrates are also provided.


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  1. SPECIAL STAINS DR HUZLINDA HUSSIN DEPT OF PATHOLOGY 13 MARCH 2018

  2. H&E stain is routine stain. - It is the preliminary or the first stain applied to the tissue sections - Gives diagnostic information in most cases. A special stain is a staining technique to highlight various individual tissue component once we have preliminary information from the H&E stain

  3. CLASSIFICATION: 1. Stains forcarbohydrates 2. Stains for amyloid 3. Nucleic acidstains 4. Lipid stains 5. Stains formicroorganisms 6. Connective tissuestains 7. Stains for pigments andminerals

  4. CARBOHYDRATES SIMPLE CARBOHYDRATES (molecules composed purely of carbohydrates) GLYCOCONJUGATES (molecules composed of carbohydrates and other molecules suchas protein and lipid) Monosaccharides (glucose,mannose,galactose) Proteoglycans Oligosaccharides (sucrose,maltose) Mucins Others glycoproteins Polysaccharides (glycogen,starch)

  5. 1. STAINS FOR CARBOHYDRATE Periodic acid schiff(PAS) technique 1. Alcian blue method 2. Combined alcian blue- PAS techhnique 3. Mucicarmine technique 4. Colloidal iron technique 5. Metachromatic staining Iodine staining for glycogen 6. 7. Enzymatic digestion technique Diastase digestion, Sialidase digestion, Hyaluronidase digestion 8.

  6. CARBOHYDRATE STAINING Components stained Possible uses & comments 1. Periodic acid Schiff (PAS) Glycogen Starch Mucin (neutral mucin; GI tract, prostate and acid-simple non-sulfated and acid- complex sulfated mucins) Basement Membrane Alpha Anti Trypsin Reticulin Fungi(capsules) Pancreatic Zymogen Granules Thyroid Colloid Corpora Amylacea Russell Bodies Glycogen storage disorder. Basement membrane in glomerular disease Staining macrophages in Whipple's disease Mucins in adenocarcinoma of large intestine Demonstration of fungi Classification of tumours with glycogen(e.g.Seminoma, rhabdomyosarcoma,ewing s/ PNET, renal cell carcinoma). Spironolactone bodies. tissue can be pre-digested with diastase to remove glycogen (PAS-diastase). Some mucins are PASD (PAS with predigestion with diastase) positive (i.e. stain is present after diastase predigestion; also called diastase resistant); glycogen is PASD negative (also called diastase sensitive because diastase removes PAS staining) **stains MAGENTA/ RED Nuclei: BLUE

  7. Candida in lung, PAS stain. Glycogen in Ewing's sarcoma, PAS stain This is nodular glomerulosclerosis involving a glomerulus in the kidney, as seen with the PAS stain.

  8. Note PAS +ve cytoplasmic granules partly resistant to diastase

  9. CARBOHYDRATE STAINING Components stained Possible uses & comments The pH of this stain can be adjusted to give more specificity. 2. Alcian blue Acid mucins BLUE Acid-simple non-sulfated: Contain sialic acid Found in epithelium (gallbladder [benign, adenocarcinoma], intestinal metaplasia in stomach) Positive for PAS, Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes They resist hyaluronidase digestion Acid-simple mesenchymal: Contain hyaluronic acid and digest with hyaluronic acid Found in tissue stroma and sarcomas Positive for Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes Negative for PAS Acid-complex sulfated: Found in adenocarcinomas Usually positive for PAS, Alcian blue at pH 1, colloidal iron, mucicarmine, and metachromatic stains They resist hyaluronidase digestion Acid-complex connective tissue: Found in tissue stroma, cartilage, and bone Includes chondroitin sulfate, keratan sulfate Positive for Alcian blue at pH 0.5 Negative for PAS Nuclei- RED Cytoplasm- PINK This is a combined method utilising the properties of both the PAS and Alcian blue methods to demonstrate the full complement of tissue proteoglycans. demonstrates both acidic- neutral- and mixtures of acidic and neutral mucins. 3. Alcian blue/ PAS The rationale of the technique is that by: First staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique. Those neutral mucins which are solely PAS positive will subsequently be demonstrated in a contrasting manner. Where mixtures occur, the resultant colour will depend upon the dominant moiety. Results acidic mucins ............................... blue neutral mucins ............................. magenta mixtures of above ......................... blue/purple nuclei ....................................... deep blue

  10. Alcian blue/ PAS stain Acid mucins - blue. Neutral mucins - magenta. Mixtures - purple. Nuclei - grey/blue. The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

  11. CARBOHYDRATE STAINING Components stained Possible uses & comments Mucicarmine is the most specific stain for mucin (very specific for epithelial mucin), but it is very insensitive. Useful for identification of adenocarcinoma ( especially of GIT). Capsule of fungus cryptococcus neoformans is also detected 4. Mucicarmine Mucin/ capsule of cryptococcus deep rose to red Nuclei black Other tissue elements light yellow CRYPTOCOCCUS STAINED BY MUCICARMINE The cytoplasm of the cells lining this neoplastic gland in a colonic adenocarcinoma are pink with the mucicarmine stain, indicative of mucin production typical for an adenocarcinoma at this site

  12. 2. STAIN FOR AMYLOID I. Congo red -Also called amyloid stain -Must examine stained tissue with standard and polarized light -Amyloid under polarized light has apple green birefringence, based on the molecule being in an antiparallel beta-pleated sheet. - amyloid-red; nuclei- blue

  13. AMYLOID Extracellular , amorphous , eosinophilic material Composed of protein in an antiparallel - pleated sheet configuration In H&E stain , can be confused with hyaline and fibrinoid substances Earliest special stain used for amyloid was Iodine by Virchow

  14. Amyloid displays apple-green birefringence under polarized light. AMYLOID BY CONGO RED STAIN UNDER LIGHT MICROSCOPE

  15. 3. Stains for lipid Oil Red O Sudan black B I. II.

  16. LIPIDS SIMPLELIPIDS COMPOUND LIPIDS DERIVEDLIPIDS - FATS - OILS - WAXES -Derived from simple or compound lipids by hydrolysis -cholesterol -Bile acids - c/o fatty acids, alcohol and one more group such as phosphorus or nitrogen

  17. Simple lipid is best demonstrated with fresh frozen sections Best fixative Formal calcium (2% calcium acetate + 10% formalin)

  18. I. Oil Red O Stain identifies neutral lipids and fatty acids Fresh smears / cryostat sections of tissue are necessary because alcohols used in tissue processing remove lipids Rapid and simple routine stain Uses: Differentiate fibroma from thecoma (not that important a distinction) Diagnose renal cell carcinoma, sebaceous gland tumors of skin, lipid-rich carcinomas Identify fat emboli in lung tissue or clot sections of peripheral blood

  19. Oil red O stain of fat emboli in lung.

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