Special staining of carbohydrates

1. Carbohydrates
Mr:Mahmoud Ibrahim Osman

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

3. – Carbohydrates are defined chemically as ketone or
aldehyde derivatives of polyhydroxy alcohols, and
they may be classified as monosaccharides
(mono:1 saccharide: sugar unit) oligosaccharides
(oligo: few, eg, 2-10), or polysaccharides (poly:
many).

4. – Glucose is the only monosaccharide found in
the body in any demonstrable quantity;
however, because glucose is extremely
soluble in aqueous solution and is of small
molecular size, it cannot be demonstrated in
tissue sections.

5. – For the same reasons, oligosaccharides also
cannot be demonstrated in tissue sections.
Glycogen, a polymer of glucose, is the form in
which carbohydrates are stored in humans, with the
liver and skeletal muscles serving as the primary
storage sites.(Home work: other sited which
glycogen is found)

6. – Glycogen is relatively insoluble in aqueous
solutions and thus can be demonstrated in
tissue sections. The remainder of the
carbohydrates with which we are concerned
is conjugated to either protein or lipid.

7. Culling system for polysaccharides
classification
– Culling used a system that placed the naturally
occurring polysaccharides in 4 groups based on
histochemical differences.
– GROUP 1: NEUTRAL POLYSACCHARIDES
(NONIONIC HOMOGLYCANS):
– 1. Glucose-containing: glycogen, starch, cellulose

8. – 2. N-acetyl-glucosamine-containing: chitin
– This group gives a very positive PAS reaction and a
negative reaction with the other frequently used
carbohydrate stains (alcian blue, colloidal iron,
mucicarmine)

9. – GROUP II: ACID MUCOPOLYSACCHARIDES
(ANIONIC HETEROGLYCANS):
– 1. Carboxylated (COOH): hyaluronic acid, found in
connective tissues and umbilical cord
– 2. Sulfated(OS03 H) and carboxylated (COOH)
– Chondroitin sulfate A (chondroitin-4-sulfate)

10. – Chondroitin sulfate C (chondroitin-6-sulfate), found in
cartilage, chondrosarcomas, cornea, and blood vessels
– Chondroitin sulfate B (dermatan sulfate), found
principally in skin, also in connective tissue, aorta, and
lung
– Heparin, found in mast cells and the intima of arteries

11. – 3. Sulfated only (COOH-free): human aorta and
bovine cornea.
– All polysaccharides in this group are acidic (anionic)
and are thought to be attached to protein, even
though the word protein does not appear in the
name.

12. – The acid mucopolysaccharides are the so-called
connective tissue mucins and are PAS negative, but
stain with alcian blue, colloidal iron, and
mucicarmine.

13. – GROUP III: GLYCOPROTEINS (MUCINS, MUCOID,
MUCOPROTEIN, MUCOSUBSTANCES):
– 1. Neutral: mucin in stomach, Paneth cell granules
– 2. Carboxylated (COOH): sialoglycoproteins that contain sialic
acid but no sulfate
– Sialomucins found in submaxillary gland mucin, small
intestine mucins, fetal mucins, the upper part of colonic
crypts, and human sublingual gland

14. • Serum glycoproteins
• Blood group substances
These are mostly epithelial mucins, but some may
occur in connective tissue. These glycoproteins are
potentially but not necessarily, PAS positive.

15. – GROUP IV: GLYCOLIPIDS:
– 1. Cerebrosides: fatty residue bound to a carbohydrate
structure .
– 2. Phosphatides: PAS-positive, noncarbohydrate-
containing lipids, including lecithin, cephalin, and
sphingomyelin. This compound is included because of
PAS positivity.

16. – The term acid mucosubstances's sometimes used
to include both the acid mucopolysaccharide and
the .acidic glycoproteins and although both
groups react similarly with many of the
histochemical techniques (eg, alcian blue), they
may react differently with others (eg, PAS)

17. Special Staining Techniques
– PAS REACTION (McManus 1948, CARSON 1983]
– Purpose
– Demonstration of polysaccharides( Glycogen)
and neutral mucosubstances.

18. – Principle:
– The reaction is based on the oxidation of certain
tissue element to aldehyde~ by periodic acid. The
most common reactive group is the l, 2 glycol
group, but other groups are also selectively
oxidized by periodate

19. – Fixative
– 10% neutral-buffered formalin or Bouin solution. Blood
smear should be fixed in methyl alcohol for 10 to 15
minutes.
– Quality control:
– Kidney is most sensitive control section. If the technique
used to demonstrate glycogen used section of liver.

20. – Reagents:
– Periodic acid 0.5%
– Schiff reagent
– Mayer's Hematoxylin

21. – TEST FOR QUALITY OF SCHIFF REAGENT:
– Place 10 ml of formalin in beaker and add few drops
from Schiff reagent If the solution rapidly turns reddish
purple, it is good: If t he reaction is delayed and the
– resultant colors a deep blue-purple the solution is
breaking down

22. – Result:
– Glycogen_, neutral mucosubstances;, certain
epithelial sulfoimucins and sialomucins, colloid
material of the thyroid: and pars intermedia of
the pituitary
– basement membrane, (PAS positive) bright rose

23. Glycogen stain by PAS

24. Neutral Mucin stain by PAS

25. Technical Notes
– Reid and Culling [1980] state that, contrary to the
generally held assumption, Schiff staining after
periodate oxidation does not necessarily indicate the
presence of carbohydrate residues, , and conversely, the
absence of staining does not necessarily mean that
carbohydrate residues are absent.

26. – They concluded that the intensity of staining in the
routine PAS reaction is the result of a combination of 4
factors.
– a. number of the available 1, 2 glycol groups
– b. reactivity of Schiff reagent with the reaction product
– c. structure of the polymer oxidizes
– d. exact procedural reaction condition!

27. – For color development, washing in tap water is very
important after the staining by Schiff reagent.
– Glutaraldehyde is not recommended as a fixative if PAS
reaction are to be performed. Glutaraldehyde is a dialdehyde.
– Chromate-containing fixative may over oxidize reactive
groups during fixation, and the resulting reaction with Schiff
reagent maybe weak

28. – Oxidizing agents other than periodic acid have also
been used before the Schiff reagent (eg, chromic and
potassium permanganate), but these other reagents are
stronger oxidizers than periodic acid and will oxidize
many groups beyond the reactive aldehyde stage.

29. PAS REACTION WITH DIASTASE
DIGESTION [LUNA 1968]
– Purpose
– Demonstration of glycogen in tissue sections
– • Principle
– This is a very sensitive histochemical method for glycogen.
Diastase and alpha-amylase act on glycogen to depolymerize
it into smaller sugar units (maltose and glucose) that are
washed out of the section.
– The Schiff reaction has been described in the PAS procedure.

30. – Fixative
– 10% neutral-buffered formalin, formalin alcohol, or
absolute alcohol.
– Quality Control
– 2 control sections of liver containing glycogen must be
used, 1 labeled "with" and the other labeled "without."
Cervix (including both endocervix and ectocervix) is
also an excellent control.

31. – Reagents:
– Periodic acid 0.5%
– Schiff reagent
– Mayer's Hematoxylin
– Malt Diastase Solution

33. Technical Notes
– Malt diastase, containing both alpha and Beta amylase, is
commonly used for digestion but tends to loosen the
sections and does not always completely digest the
glycogen. For this reason ,as well as the decreased
digestion time, many histotechnologists prefer to use
human saliva, which contains only alpha-amylase. If
preferred, digest with saliva for 20 minutes at room
temperature.

34. – Glycogen fixed in picric acid-containing fixatives may
be more resistant to diastase digestion than when
digestion follows other fixatives

35. BEST CARMINE [SHEEHAN 1980;
LUNA 1992
– Purpose
– Demonstration of glycogen
– Principle
– The Best carmine technique for glycogen is not as
specific as the PAS method with and without diastase
digestion and it does not demonstrate as much glycogen.
The stain binding with glycogen by hydrogen bond.

36. – Fixative
– Absolute alcohol is preferred; Carnoy and Bouin solution may also
be used.
– Quality Control
– A section of liver should be used.
– Reagents
– Carmine solution
– Differentiating Solution( Ethanol+Methanol+DW)

38. Technical Notes
– Vacca [1985] states that because the PAS reaction is
more pleasant to handle than the ammoniacal solutions
of the Best carmine method, is more specific, and is
chemically well understood, it is the method of choice
for glycogen.
– Autopsy liver sections are frequently depleted of
glycogen. If possible, use a surgically removed liver for
obtaining control sections.

39. 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.

40. 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.

41. – 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

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

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

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

45. MAYER MUCICARMINE (MALLORY
1942; PAYNE 1981]
– Purpose
– Staining of "epithelial" mucin in tissue sections .
– Principle
– In the past, this was thought to be primarily an empirical
stain; however, the specificity of the mucicarmine stain
was compared with 8 other techniques for mucin by
– Lauren and Sorvari [1969], and the staining pattern was
comparable to that of alcian blue.

46. – It stains carboxylated and sulfated mucins, but not
neutral mucins. Aluminum is believed to form a chelation
complex with the carmine; the resulting compound has a
net positive charge and attaches to the acid groups of
mucin.
– Fixative
– 10% neutral-buffered formalin

47. – Quality Control
– Section of un autolyzed colon, small intestine, or
appendix
– Reagents
– Mucicarmine stock Solution
– Mucicarmine working Solution
– Weigert Iron Hematoxylin
– Metanil Yellow, 0.25% Solution

49. Technical Notes
– If Gill hematoxylin is used as the nuclear stain, the mucin
may have a bluish cast.
– Mucin is a term used to describe the intracellular
secretions of various cells, and although these secretions
appear to be microscopically similar, they differ slightly
in composition.
– Culling lists the following properties of mucin

50. – staining with basic dyes
– Metachromatic
– precipitated by acetic acid (except gastric mucin)
– soluble in alkaline solutions

51. ALCIAN BLUE, pH 2.5
– Purpose
– Demonstration of acid mucopolysaccharides (popular
method for acid mucin why home work)
– Principle
– Alcian blue is a copper phthalocyanin basic dye that is
water soluble and colored blue because of its copper
content. When used in a 3% acetic acid solution (pH 2.5),

52. – alcian blue stains both sulfated and carboxylated acid
mucopolysaccharides and sulfated and carboxylated
sialomucins (glycoproteins). Alcian blue is believed to
form salt linkages with the acid groups of acid
mucopolysaccharides.
– Fixative
– 10% neutral-buffered formalin or Bouin solution

53. – Quality Control
– A section of unautolyzed small intestine, appendix, or
colon should be used as a positive control.
– Reagents
– Alcian blue (ph 2.5)
– Nuclear-Fast Red Solution

54. – Results
– Weakly acidic sulfated mucosubstances Dark blue
– Hyaluronic acid Dark blue
– Sialomucins Dark blue
– Background Pink to red
– Nuclei Red

56. Technical Notes
– Complete hydration of the sections during the
deparaffinization and hydration steps is very important
for alcian blue staining, because some alcianophilic
structures hydrate slowly; if not completely hydrated, the
result will be weak staining.
– Prolonged staining in the alcian blue solution may cause
nuclear staining

57. – If the slides are not washed well with water after the
nuclear fast red, clouding will result when the slides are
placed in the alcohols

58. ALCIAN BLUE, pH 1.0
– Purpose
– Demonstration of sulfated mucosubstances
– Principle
– When used in a O.lN hydrochloric acid solution (pH 1.0),
alcian blue stains only sulfated acid mucopolysaccharides and
sulfated sialomucins (glycoproteins). Acid
mucopolysaccharides and sialo mucins that are carboxylated
only will not be stained

59. – Quality Control
– A section of unautolyzed small intestine, appendix, or
colon should be used as a positive control
– Reagents
– I% Alcian Blue Solution, pH 1.0
– Fast red neuclar stain

60. – Results
– Sulfated mucosubstances Pale blue
– Background Pink to red
– Nuclei Red

62. ALCIAN BLUE WITH
HYALURONIDASE
– Purpose
– Differentiation of epithelial and connective tissue mucin
– Principle
– The alcian blue reaction Staining will disappear or be dramatically
reduced when tissue sections containing hyaluronic acid,
chondroitin sulfate A, or chondroitin sulfate C ("connective tissue"
mucin) are digested with testicular hyaluronidase. Glycoproteins
("epithelial" mucins) will not be affected.

63. – Fixative
– 10% neutral-buffered formalin is preferred
– Reagents
– Hyaluronidase Digestion Solution
– Buffer Solution, pH 6.0
– Alcian Blue Staining Solution
– Nuclear-Fast Red Solution

64. – Results
– Without digestion , acid mucopolysaccharides and sialomucins
Deep blue
– With digestion mucosubstances containing hyaluronic acid and
chondroitin sulfates A and C: Marked loss of staining

65. ALCIAN BLUE-PAS
– Purpose
– Differentiation between neutral and acidic mucosubstances; this
procedure is used in many laboratories today for the detection of
intestinal metaplasia.
– Principle
– Acidic mucosubstances are stained with the alcian blue technique
and neutral mucosubstances are stained by the PAS reaction.

66. – Quality Control
– Use a kidney or a mucin control, depending on the diagnostic tissue
to be stained. A section of cervix containing both endo cervix and
ectocervix also provides a good control.
– Reagents
– Alcian blue ph. 2.5
– Periodic acid
– Schiff reagent

67. – Results
– Exclusively acid mucosubstances
Blue
– Neutral polysaccharides
Magenta
– Certain substances will be colored by both PAS and alcian
blue Purple

69. 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.

70. Separate different types of acid mucin, each
type has CEC as following:
0.06M magnesium chloride-all acid mucin
stain
0.2-0.3 M --- only sulfate mucin stain blue

71. 0,5-0.6 M ---only strongly sulfated stain blue
0.7-0.8 M ---heparin/heparan stain blue

72. MULLER-MOWRY COLLOIDAL
IRON
– Purpose
– Demonstration of carboxylated and sulfated mucopolysaccharides
and glycoproteins
– Principle
– Colloidal ferric ions are, at a low pH, absorbed principally by
carboxylated and sulfated mucosubstances
– Reagents
– Ferric Chloride, 29% Solution

73. – Working Colloidal Iron Solution
– Potassium Ferrocyanide Solution, 2% Solution
– Ferrocyanide-Hydrochloric Acid Solution
– Nuclear-Fast Red Solution

74. – Results
– Acid mucopolysaccharides and sialomucins Deep blue
– Nuclei Pink-red
– Cytoplasm Pink

76. Technical Notes
– Strongly acid mucins that do not stain with alcian blue also do not
stain with colloidal iron
– The PAS stain can be used as a counterstain for this procedure also.
PAS-positive material will be magenta, mixtures of neutral and
acidic mucosubstances will be purple, and acidic mucosubstances
will be blue.

77. – This method usually gives a more intense color than
alcian blue, but colloidal iron is not considered specific
for acid mucopolysaccharides
– This method is excellent for the demonstration of
Cryptococcus neoformans
-.

78. Some background staining may be seen in some section
because of the presence of connective tissue mucin;
however, if strong background staining noted, the stain
should be repeated

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

80. 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.

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

82. Diagnostic applications:
– 1/ To diagnose poorly differentiated adenocarcinoma.
– 2/ Diagnostic significant in certain situation :
– * Hyaluronic acid –follicular mucinosis/ -myxoid
liposarcoma/-mesothelioma
– Neutral mucin- carcinoma of stomach

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

84. 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

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

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