This document provides an overview of carbohydrates, including their classification, structures, functions, and histological staining properties. Carbohydrates are classified as simple carbohydrates or glycoconjugates. Glycogen and mucin are two important carbohydrates for histological analysis. Glycogen stains with PAS, Best's carmine, and other techniques. Mucins include acid and neutral forms that stain differently with Alcian blue, PAS, and other histochemical stains depending on their composition. Carbohydrates play important roles in cellular metabolism and structure.

1. Carbohydrates
Mahmoud Ibrahim Osman
MSc in Histopathology and Cytology

2. Introduction
Carbohydrates- ( hydrated carbon)
are important organic compounds
that include sugars, starch, cellulose
and polymers that are mostly linked
to proteins.

3. The he 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:

4. protein folding, cell adhesion,
enzyme activity, and immune
recognition.

5. Classification of carbohydrates
Carbohydrates classified into two
main categories:
simple carbohydrates or those
molecules composed purely of
carbohydrates,

6. and glycoconjugates, those
molecules composed of
carbohydrates and other molecules
such as protein or lipid

7. Glycogen and Mucin are the two
main entities to be considered in
tissue carbohydrate demonstration

8. Polysaccharides
A polysaccharide is a large
macromolecule composed of
multiple monosaccharides joined by
covalent bonds referred to as
glycosidic linkages.

9. Glycogen
Glycogen is the only polysaccharide
found in animals that frequently is
evaluated by histochemical
techniques.

10. Simple polysaccharide with consists
of branched or straight chains D-
glucose units found intra cytoplasmic
serves as a major form of stored
energy reserves in humans.

11. In EM the glycogen occur in the
following forms;
Alpha: found as rosettes forms or
clusters of beta particles measured
60-250 nm in diameter.

12. Beta: occur as free particles or as a
part of a rosettes formation 20-40nm
Gamma: non-particulate found
between beta particles in the alpha
rosettes.

13. Normally glycogen found in:
Greatest amount in: liver, cardiac and
skeletal muscles.
Significant amount in: hair follicle,
endometrial gland

14. cervical and ectocervical epithelium
Few amount in: umbilical cord,
mesothelial cells, neutrophils,
megakaryocytes

15. Fixation and Section Preparation
Fixative containing alcohol or picric
acid are favorable for glycogen
demonstration eg; bouins and
Rossman solution, 80% alcohol .

16. Aqueous fixative eg; formalin give
adequate fixation.
Suza-zenker are contraindicated.
At EM: potassium permanganate and
glutraldehyde

17. Streaming artifacts (Polarization)
One of the difficulties associated
with the fixation of glycogen.
Definition: the tendency of glycogen
to stream through the cell with the
fixative toward one pole when it
fixed at RT.

18. . Overcoming:
1.Immediately fixation.
2.Fixation should be carried out at 4c
3.Using freeze drying technique

19. Glycogen staining
Langhan’s iodine method:
Glycogen gives mahogany brown
while other tissues stain yellow.
Impermanent- nonspecific

20.  H&E:
cells containing abundant glycogen
will stain deeply compared with those
containing little(weak red).

21. Periodic Acid-Schiff Reaction
(PAS)
The PAS technique is without
question the most versatile and
widely used technique for the
demonstration of carbohydrates or
glycoconjugates.

22. The PAS technique may aid in the
differential diagnosis of tumors
through the detection of mucins or
glycogen.

23. Mechanism of the PAS
technique
The PAS stain is a histochemical
reaction in that the periodic acid
oxidizes the carbon to carbon bond
forming aldehydes which react to
the fuchsin-sulfurous acid which
form the magenta color.

24. RESULTS:
Glycogen: magenta
Nuclei : blue

26. Best's carmine
Carmine: extracted from
cochineal (insect) with water and
precipitated with alum.

27. Mechanism of Bests carmine
Hydrogen bonding formation
between OHˉ groups on the glycogen
and H atom of the carminic acid

29. Hexamine sliver technique
Give similar result to PAS reaction.
Principle:
Following chromic acid oxidation, aldehydes
are formed from glycogen, these will reduce a
hexamine silver nitrate to black compounds.

30. Enzyme control
Use to specified the techniques applied in
glycogen demonstration:
Alpha amylase: extracted from hog
pancreas and Bacillus subtilis and
Aspergillus oryzea.both branched or
straight chain glycogen are digested, these
releases glucose and maltose.

31. Beta amylase: obtained from sweet
potato digest straight chain and release
maltose alone.
Diastase: commonly used as it is; easy,
stable, cheep, extracted from malt and
contain both alpha and beta amylase

32. Saliva: highly effective.
Pectinase: effective with abnormal
glycogen. Found in certain
glycogenesis, which is not digested
easy by amylase

34. Medical importance
Some glycogen storage diseases :
A-von Gierke disease (deficiency of
Glucose-6-phosphatase)
B-Pompe disease(deficiency of Acid
maltase)

35. Also demonstration of glycogen can
help in diagnosis of carcinomas of
Bladder,Kidney,Liver,ovary and
adenocarcinoma of
Pancrease,Lung,Seminoma
Mesothelioma

36. Mucin
High molecular weight glycolized
protein secreted by specialized
epithelial cells and connective tissue
cells

37. The carbohydrate content of a mucin
may account for up to 90% of its
molecular weight.

38. In contrast to the
glycosaminoglycan's, which are
strongly acidic polyanions, the
polysaccharide chains of the mucins
vary from neutral or weakly acidic to
strongly acidic.

39. mucoid carbohydrates can be
divided into three main types:
1.Mucopolysaccharides
2.Mucoprotein
3.Mucolipids

40. Mucoprotein
Predominantly protein and contains
more than 4% hexosamine , when
the polysaccharides over 20% known
as sialomucin ,

41. when the polysaccharides less than 4%
we called glycoprotein and classified
with mucoprotein and can not be
distinguished from them . PAS---positive

42. Glycolipids
Made up of fatty acids combined
with carbohydrates , usually
galactose the cerebrosides are the
most important glycolipids PAS---
positive

43. Functions of mucins
1.Lubricator function
2.Environment for ionic and molecular
diffusion
3.Cell to cell adhesion with specific cell
surface

44. Protective role (innate immunity) from
microbes and chemical and mechanical
agents throughout GIT, Respiratory
tract ,reproductive system .

45. Mucopolysaccharides(Mucin)
Polysaccharides-protein complexes
divided into:
- acid mucopolysaccharides
- and neutral mucopolysaccharides

46. Neutral mucopolysaccharides
carbohydrate made up of hexose
units usually acetylated such as
glucosamine combined with proteins
and calcium.

47. present in alimentary ,respiratory
tract , prostate gland ,PAS----positive

48. Acid mucopolysaccharides
composed of chains of glucosamine
and glucuronic acid and proteins
Divided into

49. 1.Simple acid mucopolysaccharides;
Contain carboxylated glucose units
(glucuronic acid) e g (hyaluronic
acid )

50. 2. Complex acid mucopolysaccharides:
In addition to glucuronic acid –sulfated
glucosamine units are includes e g chondroitin
which occur in cartilages and connective tissues
also heparin and heparan are others examples

51. Acid mucin : divided into sulfated
and carboxylated mucin
Sulfated mucin: divided into
strongly and weakly

52. Strongly sulfated mucin: divide into
strongly sulfated epithelial and strongly
sulfated connective tissue mucin
(proteoglycan )

53. Strongly sulfated CT mucin
Strongly sulfated CT mucin:
 Highly sulfated substances produced by
fibroblast ,endothelial, osteocytes ,
chondrocytes ,mast cells , react at low PH
with cationic dyes and PAS negative.

54.  The following types
1. Chondroitin sulfate A : found in
cartilage
2. C,S,B: found in aorta , heart valves ,
dermis of skin.

55. 3. C,S,C : in cartilage ,umbilical cord ,
dermis of skin.
4- Heparin/heparan sulfate: heparan found
in aorta , cardiac CT, and heparin in mast
cells.

56. 5-Keratan sulphate: present in
cornea, ageing cartilage
6-Hyaluronosulphate: found in
cornea

57. Strongly sulfated epithelial mucin :
 in bronchial glands and in minor
fraction in intestinal goblet cell ,
histologically react at low PH.

58. But differ from that of connective
tissue mucin in being PAS positive

59. Weakly sulfated epithelial mucin
(sulfomucin) : epithelial in type,
present in colonic goblet cell , react
at slightly higher PH

60. Carboxylated mucins :
Sialomucin: contain derivatives of
neuromeric acid (sialic ) epithelial in
origin, divide into:

61. a-Enzyme labile ( N-acetyl sialomucin):
digested by sialidase , present in
bronchial sub mucous gland , sub
mandibular salivary gland ,

62. goblet of small intestine ,PAS--
positive

63. b. Enzyme –resistant (N-acetyl -O-
acetyl neuromeric acid ) sialidase
resistant , found in mucosa of large
intestine –stomach-bronchus , PAS----
negative

64. 2- Hyaluronic acid: occur in CT
formed by fibroblast ,important
constituent of synovial fluid also
found in umbilical cord ,

65. cardiac CT , dermis of skin , aorta ,
bone and cartilages

66. Diagnostic applications:
1/ To diagnose poorly differentiated
adenocarcinoma.
2/ Diagnostic significant in certain
situation :

67. * Hyaluronic acid –follicular
mucinosis/ -myxoid liposarcoma/-
mesothelioma
Neutral mucin- carcinoma of
stomach

68. Sialomucin (N-acetyl form)-
Transitional epithelium of
colon/rectum
Sialomucin (O-acetyl form )-ca of
colon/rectum

69. Demonstration of mucins
Aqueous fixatives (formalin) are
satisfactory.
Muco polysaccharide are better
preserved in alcoholic fixatives

70. Hyaluronic acid when occur free is better
fixed in 10% formal-alcohol or 10 %
formal sublimate.
Other fixatives 2% calcium acetate in
10%formalin

71. Mucin staining
1/ H&E:
 Mucin take up the eosin ,unless
Ehrlich Hematoxylin is used where
acid mucin stains blue.

72. 2/ PAS: Mucin containing a reactive
hexose component will be PAS
positive, these include:
1. Neutral mucin
2. N acetyl sialomucin

73. 1. Epithelial sulphated mucin
2. Mucoprotein
3. Glycolipid

74. 3/ Alcian blue
Alcian blue is the highest molecular
weight (cationic dyes) formed
electrostatic bonds( salt linkage)

75.  with tissue polyanions bearing either
carboxyl or sulfate groups

76.  Alcian blue is a popular dye for
demonstrating acid mucin why?
1. Specificity
2. Strong coloration

77. 1. In solubility of staining
2. Permanence of results

78. Alcian blue using varying PH :
At PH 2.5 both sulfate and
carboxylate mucins will stain.

79. Strongly sulfate; react with alcian at
low PH (1) or less
Weakly sulfated ; will stain well
from PH2.5 , down to 1 PH

80. Hyaluronic acid and N-acetyl
sialomucin; PH (1.7-3.2 )
N-acetyl –O- acetyl sialomucin ; PH
1.5

81. Alcian blue involving critical
electrolyte concentration(CEC)
CEC is point at which the amount of
electrolyte ,such as magnesium
chloride, in alcian blue solution is
sufficient to prevent staining.

82. Separate different types of acid
mucin, each type has CEC as
following:

83. 0.06M magnesium chloride-all acid
mucin stain
0.2-0.3 M ---only weakly and
strongly sulfate ,stain blue

84. 0,5-0.6 M ---only strongly sulfated
stain blue
0.7-0.8 M ---heparin/heparan stain
blue

85. 4/ Dialyzed iron –prussian blue
tech.
This is another popular technique for
demonstration acid mucins.
At low PH colloidal iron will be
adsorbed onto tissue polyanions (
sulphate , carboxylate group)

86. and subsequently ,visualized by
conversion to ferric ferrocyanide
using (Perl's tech)
It is more sensitive ,but complex

87. 5/ Southgate mucicarmine tech-
The rationale : not fully understood
,the aluminum/carmine compound
appear to be positively charged and
combined with negatively charged
acid mucosubstances.

88. Results :
Mucins :Red
Nuclei : Blue

89. 6/Metachromatic staining
method
Acid mucosubstance can be
demonstrated by numbers of
metachromatic dyes.
The most useful dye is azure A

90. Sulfated and carboxylated mucins
are metachromatic while neutral
mucin is orthochromatic.
Sulfated mucin at PH3 and
carboxylated at PH5

91. Combined alcian blue PAS methods
Acid mucins and neutral mucins are
separated , also useful to demonstrate
any mucins . First all acid mucin
stained with alcian blue leaving only
neutral mucin to be demonstrated by
PAS reaction.

92. Alcian blue-Alcian yellow
Used to distinguished b/w sulphate and
carboxylate, involving initial staining
with low Ph. alcian blue, followed high
Ph. alcian yellow
Result :
sulphate mucin- blue
carboxylate mucin -yellow

93. Aldehyde fuchsin –Alcian blue
technique
Separate sulphate from carboxylate.
The rationale:
Depends on the greater affinity of
aldehyde fuchsin for sulphate mucin .

94. so that by first staining with this
solution sulphate stained purple
,rendering carboxylated mucin to be
stain by alcian blue (counterstaining

95. Blocking tech and Enzyme
controls
Improved specificity, by enzyme
action or by blocking staining
(chemical action )

96. Blocking
Methylation:
using hydrochloric acid in methyl alcohol for
blocking the staining reaction of carboxylated
mucin by esterification of carboxyl group and
sulphate group by desulphation.

97. Methylation and Saponification:
After Methylation ,saponification with
potassium hydroxide in ethyl alcohol ,
will restore the staining of carboxyl
groups but sulphate groups still blocked.

98. Sialidase digestion:
useful method for identifying a certain
type of sialomucin.

99. Other types of carbohydrates
Chitin:
This is hyaline substance wide
distributed in non human tissue for
example exoskeleton of insects.

100. In human only seen lining the wall of
hydatid cyst of lung or liver due to
infestation of larvae of dog tapeworm
Echinococcus granulosus.
PAS positive Diastase resistance

101. Starch
Can be found in tissue as
contaminant from surgical gloves
powder.
PAS positive with iodine pale blue-
Diastase labile

102. Cellulose
Seen in undigested food specially in
the section taken from GIT.
PAS positive brown color with
iodine