Demonstration of carbohydrates in histopatholgy

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