CARBOHYDRATE STAINS

INTRODUCTION
 Glycobiology- a complex discipline that permeates
diverse fields as cell biology, microbiology, enzymology,
and molecular biology
 Histochemical techniques are used for the detection and
characterization of carbohydrates and carbohydrate
containing macromolecules (glycoconjugates)
 Provide invaluable information which may aid the pathologist in
diagnosing and characterizing various pathological conditions
including neoplasia, inflmmation, autoimmune disorders and
infectious diseases
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BASIC CLASSIFICATION OF
CARBOHYDRATES
 Monosaccharides
 Glucose
 Mannose
 Galactose
 Oligosaccharides
 Sucrose
 maltose
 Polysaccharides
 Glycogen
 Starch
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BASIC CLASSIFICATION OF
GLYCOCONJUGATES
 Connective tissue  Other glycoproteins
glycoconjugates  Membrane proteins
 Proteoglycans (receptors, cell adhesion
molecules)
 Hyaluronic acid
 Blood group antigens
 Mucins
 Glycolipids
 Neutral mucins
 Cerebrosides
 Sialomucins
 gangliosides
 Sulfomucins

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SUMMARY OF DIFFERENT TYPES OF
CARBOHYDRATES AND GLYCOCONJUGATES
TYPE LOCATION FUNCTION ASSOCIATED PATHOLOGICAL
CONDITION
Glycogen Liver, skeletal Storage form of Found in wide range of
muscle, cardiac carbohydrate malignancies- Ewing’s
muscle, hair sarcoma,
follicles, cervical rhabdomyosarcoma,
epithelium seminoma, glycogen storage
disease
Proteoglycans and Cartilage, heart Support, Found in myxoid
hyaluronic acid valves, blood lubrication, cell chondrosarcomas, myxoid
vessels, tendons, adhesion, etc. liposarcomas, myxoid fibrous
ligaments, ECM, histiocytomas, and in
and membranes of patients with
many cell types mucopolysaccharidoses

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SUMMARY OF DIFFERENT TYPES OF CARBOHYDRATES
AND GLYCOCONJUGATES (continued)
TYPE LOCATION FUNCTION ASSOCIATED PATHOLOGICAL
CONDITION
Mucins Epithelia of the GIT, Secreted mucins- Frequently found in
repiratory tract and lubrication and adenocarcinomas of the GIT
reproductive tract protection
Aberrant or inappropriate
Membrane-bound expression of specific mucin
mucins- cell types occurs frequently in
adhesion and neoplastic process
regulation of
proliferation
Glycoproteins Cell membrane Multiple and diverse Aberrant expression of blood
functions such as group antigens in various
Blood group antigens cell adhesion, malignancies
immune
Secreted products recognition,
(peptide hormones and regulation of
immunoglobulins) receptor ligand
binding ,etc.
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PROTEOGLYCANS
 Referred to as connective tissue mucins or
mucopolysaccharides
 Found in the connective tissues ECM and act in stabilizing
and supporting fibrous elements of connective tissue
 Highly glycosylated (90-95% is carbohydrate)
 Carbohydrated component: GAGs
 Composed of repeating disaccharides
 Ionized and carry a negative charge
 Most abundant type: chondroitin sulfates
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CHARACTERIZATION OF
GLYCOSAMINOGLYCANs (GAGs)
Glycosaminoglycans Disaccharide repeat Location
Glucoronic acid and N- Cartilage, tendons, ligaments,
Chondroitin sulfate
acetylglucosamine aorta, cell membranes
Iduronic acid and N- Skin, blood vessels, heart
Dermatan sulfate
acetylglucosamine valves
Galactose and N-
Keratan sulfate Cartilage, cornea
acetylglucosamine
Glucoronic acid and N-
Blood vessels, aorta, cell
Heparin sulfate acetylglucosamine or N- sulfate
membranes
lglucosamine
Glucoronic acid and N-
Heparin acetylglucosamine or N- sulfate Mast cell granules
lglucosamine
Synovial fluid, humor, loose
Glucoronic acid and N-
Hyaluronic acid connective tissues, small
acetylglucosamine
amount in cartilage

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MUCINS
 Consist of polysaccharide chains covalently linked to a
protein core
 Defining structure of epithelial mucins is the presence of
tandemly repeated amino acid sequences within the
protein core
 Sialic acids:
 sialisidase resitant- not detected by PAS
 sialisidase labile- clearly visible with PAS

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FIXATION
 GLYCOGEN
 Recommended fixative: alcoholic formalin
 Others: NBF, Rossman’s fluid, alcoholic formalin with picric acid
 Zenker’s-acetic acid and Susa’s are not recommended
 Fixation should be carried out in 4 C to minimize artifacts
 MUCOPOLYSACHARIDOSES
 Fresh or frozen sections are most recommended although formalin
is also satisfactory
 Typical MUCINS and PROTEOGLYCANS
 Recommended fixatives: formalin or alcoholic formalin
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TECHNIQUES FOR DEMONSTRATION
OF CARBOHYDRATES
Periodic Acid Schiff (PAS) technique High Iron Diamine
Mild PAS technique Combined High Iron Diamine and
Alcian Blue techniques (Standard- , low Alcian Blue technique
pH- , -with Varying electrolyte Combined aldehyde fuschin-alcian
concentrations, - combined with PAS) blue technique
Mucicarmine technique Metachromatic methods (ex. Azure A)
Colloidal Iron technique

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PERIODIC ACID-SCHIFF (PAS)
technique
 Most versatile and widely used technique for
demonstration of carbohydrates and glycoconjugates
 First histochemical use was by McManus for mucin
 Also used for glycogen and certain glycoproteins
 Aid in differential diagnosis of tumors through detection of
glycogen or mucins

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MECHANISM OF PAS TECHNIQUE

 PAS is based upon the reactivity of free aldehyde groups

within carbohydrates with Schiff reagent to form a bright
red magenta end product

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PAS-REACTIVE CELLS & TISSUE
COMPONENTS
 Glycogen  Fungi (capsules)
 Starch  Pancreatic zymogen
granules
 Mucin (siolomucin, neutral
mucin)  Thyroid colloid
 Basement membranes  Corpora amylacea
 Reticulin  Russel bodies

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PAS TECHNIQUE (Modified McManus)
components
 Periodic Acid solution
 Periodic acid (1g )
 Deionized or distilled water (100ml)
 Schiff reagent preparation
 Dissolve 1g basic fuschin and 1.9 g of sodium metabisulfite in 100
ml of 0.15 N HCl.
 Shake solution at intervals or with mechanical rotator for 2 hours.
Solution should be clear and yellow to light brown.
 Add 500 mg of activated charcoal and shake for 1-2 minutes
 Filter through Whatman filter paper (No. 1). Filter should be
clear and colorless.
 Store at 4C
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PAS TECHNIQUE (Modified McManus)
method
 Deparaffinize in xylene and rehydrate through graaded
ethanols
 Oxidize with Periodic acid for 5 minutes
 Rinse in several changes of deionized water
 Cover sections with Schiff reagent for 15 minutes
 Rinse in running tap water for 5-1 minutes
 Stain the nuclei with Harris’s or Mayer’s hematoxylin.
Differentiate and blue the sections
 Dehydrate, clear and mount

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PAS TECHNIQUE (Modified McManus)
results
 Glycogen, neutral/sialomucins- magenta
 Various glycoproteins- magenta
 Nuclei- blue

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PAS TECHNIQUE (Modified McManus)
notes
 For basement membranes a longer time in Periodic acid
(10 min) and Schiff reagent (20 min) may give better
result
 Post-Schiff bisulfite rinses are used for reduction of
background
 Glutaraldehyde fixatives should be avoided when using
PAS
 Glycolipid staining may be detected using frozen
sections
 Glycolipids and unsaturated lipid are significantly loss
from paraffin-embedded sections
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MILD PAS technique
 Utilizes a weak eriodic aicd oxidation step to dmeonstrate
N-acetyl sialic acid- containing mucins

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STANDARD ALCIAN BLUE technique
 Initially used for dyeing textile fibers
 Alcian blue 8GX- recommended dye for histological
techniques
 Demonstrates and precipitate hyaluronic acid, chondroitin
sulfate and heparin from an aqueous solution (pH 2.5)
 Also demonstrates acidic epithelial mucins (siolomucins &
sulfomucins) of large intestine
 Neutral mucins of gastric mucosa and Brunner’s gland are not
reactive with alcian blue

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LOW pH ALCIAN BLUE technique

 Demonstrates sulfomucins and sulfate-containing

proteoglycans which are ionized and negativley charged at
a pH 1
 Example of tissues and cell types exibiting staining with
low pH alcian blue: cartilage, goblet cell mucin of large
intestines, and mucins of bronchial serous glands

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Alcian Blue with Varying Electrolyte
Concentrations
 Uses electrolytes such as magnesium chloride
 Based on the phenomena known as critical
electrolyte concentration
 Point at which the amount of an electrolyte is
sufficient to prevent staining with alcian blue
 Time consuming and labor intensive
 But extremely useful in differentiating mucins and
proteoglycans
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COMBINED ALCIAN BLUE-PAS

 Use to differentiate neutral mucins from acidic

mucins within a tissue section
 Sections are stained with standard alcian blue (pH
2.5) method followed by PAS

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COMBINED ALCIAN BLUE-PAS
results
 Glycogen, neutral mucins, various glycoproteins-
magenta
 Acid mucins (sulfomucins and sialomucins)- blue
 Proteoglycans & hyaluronic acid- blue

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MUCICARMINE technique
 Oldest histochemical methods for the visualization of
mucins in specimens
 Remains valuable means for the demonstration of acidic
mucins
 Dye molecule used: aluminum-carminic acid comlplex
(carmine)
 Useful for identification of adenocarcinomas
 Also for demonstration of C. neoformans capsule

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Mucicarmine results
 Acidic epithelial mucins- deep rose to red
 Nuclei- black
 Other tissue elements- light yellow

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COLLOIDAL IRON technique
 initially described by Halen for detection of acid
mucopolysaccharides
 Based upon the attraction o ferric cations for the
negatively charged carboxyl and sulfate groups of acid
mucins and proteoglycans
 Tissue-bound ferric ions are visualized by treatment with
potassium ferrocyanide to form bright blue or Prussian
ble color

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COLLOIDAL IRON technique
results
 Proteoglycans, hyaluronic acid and acidic mucins-
bright blue
 Collagen-red
 Muscle and cytoplasm- yellow

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COLLOIDAL IRON technique
notes
 pH of colloidal iron is critical
 pH 2.0 or higher non-specific staining of structures other than
acidic carbohydrate will occur
 Nuclear fast red can be used as a counterstain
 Stock colloidal iron should be dialyzed to remove free acid
and unhydrolyzed iron salts

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High Iron Diamine

A useful technique by Spicer for the detection of

highly acidic sulfomucins
 Hyaluronic acid and sialomucins are not
demonstrated by this techniqui
 Facilitates the differentiation of sulfomucins from
sialomucins in tissue sections

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COMBINED HIGH IRON DIAMINE and
ALCIAN BLUE technique
 Suited for demonstration of sialomucins and
sulfomucins distribution in epithelia of the
intestines

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COMBINED HIGH IRON DIAMINE and
ALCIAN BLUE results
 Sulfated mucins and proteoglycans- black-brown
 Sialomucins and hyaluronic acid- blue
 Diamines are potentially toxic and handling should
be with great care
 Nuclear fast red may be used to enhance nuclear
contrast

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COMBINED ALDEHYDE FUSCHIN-
ALCIAN BLUE
 Used to demonstrate a wide array of tissue components
including elastic fibers, beta-cells of the islets, and
thyroid colloid, etc.

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COMBINED ALDEHYDE FUSCHIN-
ALCIAN BLUE results
 Proteogylcans and strongly acidic sulfomucins- deep
purple
 Weakly acidic sulfomucins- purple
 Sialomucins and hyaluronic acid- blue

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METACHROMATIC METHODS
 Oldest histochemical technique for identification of
charged mucins and proteoglycans
 Methylen blue, Azure A, and toluidine blue stain tissue
blue but under conditions of metachromasia they stain
tissue components purple-red
 The more acidic or highly sulfated proteoglycans the
stronger and more stable the metachromasia

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AZURE A technique results

 Acid mucins and proteoglycans- purple to red

 Tissue background- blue

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ENZYMATIC DIGESTION TECHNIQUES
•Diastase digestion
•Sialidase

•Hyaluronidase

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DIASTASE DIGESTION
 Digestion is necessary when diagnosis requires correct
identification of mucosubstance such as mucin or glycogen
 Malt diastase is frequently used for this purpose

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DIASTASE DIGESTION
procedure
 Deparaffinize two serial sections in xylene and rehydrate
through graded ethanols to water
 Place one slide in the diastase solution for 1 hour at 37C.
The other slide is an untreated control and may remain in
water for 1 hour
 Wash both slides in running tap water for 5-10 minutes
 Proceed with the PAS technique

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DIASTASE DIGESTION results

 Glycogen- bright red/magenta in untreated slide

 Glycogen- stain is absent in diastase-treated slide

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SIALIDASE
 Sialidase or neuraminidase is isolated from V.
cholerae
 Cleaves terminal sialic acid moieties from sialomucins
and glycoproteins

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SIALIDASE results
 Sialidase-labile sialic acids-bright blue in the
untreated section
 Neutral mucins- red to magenta in the untreated
sections
 Following treatment, mucins containing sialidase-
albile sialic acids stain red to magenta

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SIALIDASE notes
 Sialic acids containing an O-acetyl group usually are
resistant to sialidase

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HYALURONIDASE
 Most commonly used hyaluronidase is isolated from an
extract of bull testis
 Used for pre-treatment of specimens prior to staining with
alcian blue or colloidal iron

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HYALURONIDASE digestion
 Alcian blue (pH 2.5)
 Connective tissue proteoglycans containing chondroitin
sulfate and/or hyaluronic acid satin bright blue

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PATHOLOGIC CHANGES & DEPOSITS
FOUNDI N CONNECTIVE TISSUE
•Fibrin
•Fibrinoid
•Hyalin
•Amyloid
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FIBRIN
 Material resulting from enzymatic coagulation of plasma
globulin and fibrinogen, forming bundles which contract
into dense homogenous masses
 Substances in the blood such as hemoglobin and
glycoprpoteins become entangled with the fibrin, giving a
weak reaction when tested for hemoglobin and
glycoprotein

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Stains for demonstration of
FIBRIN
 Hematoxyln and eosin stain
 Mallory’s PTAH
 Gram Staining
 PAS

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FIBRINOID
 A mixture of exudate and altered cytoplasmic constituents
forming a homogenous eosinophilic material
 Gives the same staining reaction as fibrin
 Found in collagen disease, hypersensitivity, SLE, and
rheumatic heart disease

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HYALIN
 Refers to a wide variety of pathologic exudates and
deposits appearing in the form of a smooth homogenous
eosinophilic material in routine stains
 Seen in degenerated collagen, hypertension, atheroma,
and diabetic kidney
 No specific stain

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AMYLOID
 A semi-translucent, ground-glass or hyalin eosinophilic
substance made up of chondroitin sulfuric acid-protein complex
 Deposited in kidneys, spleen, lymph nodes, pancreas, during
chronic suppurative conditions and inflammatory conditions
 TB, leprosy, osteomyelitis
 Also found in idiopathic or primary amyloidosis in heart,
tounge, larynx, intestine, skeletal muscle, and blood vessels

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 Stains for demonstration of AMYLOID

 Gram’s iodine stain

 Amyloid- brown to blue
 Krajians amyloid stain
 Amyloid- red in clear background
 Congo red method
 Amyloid- deep pink to red color
 Metachromatic Staining
 Amyloid- purplish red or red
 Induced Fluorescence staining with thioflavine
 Amyloid- yellow fluorescence in olive-green backgorund 52

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