CARBOHYDRATE

• is used to encompass a large group

of compounds with the general
formula Cn(H2O)n. While the role of
carbohydrates in cellular metabolism
has been known for many years,
• carbohydrates have been more
recently implicated in a wide range
of cellular functions including protein
folding, cell adhesion, enzyme
activity, and immune recognition
• Histochemical techniques for the
detection and characterization of
carbohydrates and carbohydrate-
containing macromolecules
(glycocongugates) are common
practices in the histology laboratory.
• These techniques often provide
invaluable information which may
aid the pathologist in diagnosing and
characterizing various pathological
conditions including neoplasia,
inflammation, autoimmune
disorders, and infectious diseases.
 Classification of carbohydrate
• simple carbohydrates or those
molecules composed purely of
carbohydrates.
• glycoconjugates, those molecules
composed of carbohydrates and other
molecules such as protein or lipid .
• The simple carbohydrates are further
categorized as
• Monosaccharides( glucose, mannose,
galactose)
• oligosaccharides(sucrose, maltose)
• polysaccharides ( glycogen, starch )
•
• Glycoconjugates may further be broken down
into proteoglycans, mucins, and ‘other’
glycoproteins. Although lipid-carbohydrate
complexes are widely distributed in cells and
tissues.
• Glycoconjugates:
• Connective tissue
glycoconjugate(proteoglycans,
hyaluronic acid)
• Mucins( neutral mucins, sialomucins
,sulfomucins )
• Other glycoproteins( membrane
proteins (receptors, cell adhesion
molecules), blood group antigens)
• Glycolipids (cerebrosides,
gangliosides)
 Polysaccharide
• A polysaccharide is a large
macromolecule composed of
multiple monosaccharides joined by
covalent bonds referred to as
glycosidic linkages,(starch and
glycogen)
 Glycogen
• Glycogen is the only polysaccharide
found in animals that frequently is
evaluated by histochemical
techniques. Glycogen serves as a
major form of stored energy reserves
in humans.
• Carbohydrates absorbed following a
meal are converted to glycogen by
the hepatocytes of the liver. In times
of fasting, glycogen is broken down
into glucose units that can be used as
an immediate source of energy.
• Glycogen occupies a significant
volume of the cytoplasm of
hepatocytes and may even form
intranuclear inclusions.
•
• Skeletal and cardiac muscle cells,
hair follicles, endometrial gland ,
vaginal and ectocervical
epithelium ,umblical cord ,
methothelial cells , neutrophils ,
leukocytes , megakaryocyte also
store significant quantities of
glycogen.
 pathological conditions
• Abnormal accumulation in tissues of patients
with glycogen storage diseases
• Ewing’s sarcoma/PNET.
• rhabdomyosarcoma.
• Seminoma etc. Abnormal accumulation in
tissues of patients with glycogen storage
diseases
 Fixation
Fixative containing alcohols or picric
acid .(bouin`s or rossman`s solution)
neutral buffered formalin (NBF) is an
acceptable fixative for glycogen
• fixation should be carried out at 4°C
to minimize the streaming artifact
which frequently which occurs when
glycogen is fixed at room
temperature
 Techniques for the demonstration
glycogen
• Hematoxylin and eosin :-
• glycogen fail to stain with conventional
hematoxylin solution and only stain weakly , if
at all with eosin . In practice cell contain
abundant glycogen will stain weakly compared
with those containing little
 The periodic acid-Schiff (PAS)
technique:-
• Principle:-

• 1%periodic acid will bring about oxidative

cleavage of the carbon –carbon bound in

1.2 glycol or their amino or alkylamino

derivatives,to form dialdehydes,

• these aldehyde react with schiff`s
reagent give magenta color
• Alternative oxidative agent:-

• Chromic acid .

• Potassium permengnate .

• Lead tetra acetate.

• Sodium bismuthate.
• A number of methods for the synthesis
of Schiff `s reagent have been
described .All of these methods share
a common theme in the production of
an aqueous solution of sulfurous acid.
• The sulfurous acid may be generated
from the reaction of sodium
metabisulfite (Na2S2O5) with a mineral
acid such as hydrochloric acid (HCl), or
by the reaction of thionyl chloride
• the principle in preparation of
schiff`s solution involve : the
rearrangement of the chromophoric
groups present in basic fuchsin by
sulfuration.
• Any access sulfur reremaining together with

any non specific yellow dye contaminant

present in basic fuchin is remove by treatment

with activated charcol onto which the sulfur

particles are adsorbed and removed .

• Nuclear staining apply after PAS
treatment due to oxidative step
• Alum hematoxylin is popular as PAS
technique nuclear stain .
• PAS reactive cell and tissue:-
• Glycogen, starch, mucin (sialomucin,
neutral mucin), basement
membranes, α-antitrypsin reticulin,
fungi (capsules) pancreatic zymogen
granules, thyroid colloid ,corpora
amylacea ,Russell bodies.
 Enzymatic digestion techniques

• amylase or diastase techniques for

glycogen digestion are commonly
utilized in laboratories to enhance the
specificity of the PAS technique.
• Alpha amylase :-

• this is extract from hog pancrease and certain

organism such as bacillus subtiliis and
aspergillus oryzea , both branched and
straaight chain glycogens are digested by
splitting alpha 1;4glucosidic linkage .
• This release glucose and maltose ,
both of which are soluble in the
solvent used for enzyme and are thus
removed from section
• Beta amylase :-
• obtain from barely or sweet patato ,
digest straight chain glycogen only
release maltose only .
• Diastase :-
• Commenly used in glycogen digestion

• Easy to use

• Stable and cheap

• It extract from malt and contain alpha and beta amylase

• Saliva :-
• contain ptyalin (salivary amylase )
saliva high affective means of
digesting glycogen in tissue section .
• pectinase :-
• digestion of abnormal glycogen
found in certain of the glycogenoses
are not easy digested by amylase .
 Best`s carmine
• Principle:-

• The acceptable rationale is that the staining

of glycogen ia accomplished by hydrogen
bond formatiom between OH groups on the
gycogen and H atoms of the carminic acid .
• The method is highly selective rather
than specific for glycogen , as fibrin
and nutral mucin also stain.
• Potassium carbonate and potassium chloride
salts serve to inhibit non specific back
ground carmine stain due to electrostatic
bonding between the negatively charged
carmine and basic protein in tissues .
• and allow only hydrogen bond to take place
btween glycogen and carmine
• Ammonium or sodium salt can safely be
substituted for potassium salts.
• Ammonum hydroxide solution it give
ahigh pH (10-11) and there for helps
depress carmine staining of tissue
through electrostatic linkages.
 Hexamine silver techique
• Principle :
• chromic acid oxidation , aldehyde
are formed from glycogen ; this will
reduced a hexamine silver – nitrate
mixture to black compound .