Role of Histochemistry in Surgical Pathology

1. ROLE OF HISTOCHEMISTRY
IN SURGICAL PATHOLOGY
SPEAKER: DR. HAJRA K. MEHDI
MODERATOR: DR. CSBR PRASAD
SRI DEVARAJ URS MEDICAL COLLEGE, KOLAR
1

2. OUTLINE
• OBJECTIVE
• INTRODUCTION
• CLASSIFICATION
• DETAIL OF EACH STAIN
• RECENT ADVANCES
• REFERENCES
2

3. OBJECTIVE
A) Describe the different types of special staining.
B) Learn the various methods of stain demonstration.
C) Learn the procedures and diagnostic tools for each stain.
3

4. INTRODUCTION
Stains used for identification of certain structures and
chemical substances by controlled specific chemical
reaction designed to give a final color or location of the
structure or substance in the cells or tissues.
4

5. a) To confirm the diagnosis suspected on routine stain.
b) For special purposes when indicated by the clinical
situation.
• Z-N stain is ordered for Mycobacteria, Congo red stain is
requested when amyloid is suspected.
• Staining occurs when chromogen of one charge attracts
bound tissue moiety of opposite charge.
USES OF SPECIAL STAINS
5

6. BASIS OF STAINING
ACIDIC DYES BASIC DYES
Eosin Hematoxylin /Methylene blue
Carry net negative charge Carry net positive charge
React/bind with cationic components
of the cell/tissue
With anionic components of cell/tissue
Less specific (as compared with basic
dyes)
Highly pH specific
Acidophilic / Eosinophilic (cytoplasmic
filaments, intracellular membranous
components, extracellular fibers)
Basophilic substances ( Po4 of
Nucleic acids, So4 of MPS, CO
proteins) 6

7. 7

8. CLASSIFICATION OF STAINS
• CHO - GLYCOGEN
- MUCIN
• LIPIDS
• PROTEINS - FIBRIN
- AMYLOID
• CONNECTIVE TISSUE - COLLAGEN
- RETICULIN
- ELASTIC
• PIGMENT
• MICROBIOLOGY
• HAEMATOLOGY
8

9. SPECIAL STAINS FOR MUCIN
• Mucicarmine: For Neutral and Acid Mucin
• Phenylhydrazine PAS Technique: For Neutral
Mucins.
• Alcian Blue: For Acid Mucins.
9

10. MUCICARMINE
(MAYER 1896, MODIFIED BY SOUTHGATE 1927 )
• Carmine
• Aluminium hydroxide
• 50% alcohol
• Aluminium salts form a chelate compound with carmine
Results:
Mucin ---- red
Nuclei ---- blue
Applications:
1. Demonstration of epithelial mucin
2. Demonstration of encapsulated fungi (Cryptococcus)
10

11. Mucicarmine - Neutral epithelial mucins – Small intestine
11

12. Cryptococci
12

13. PAS
(MCMANUS 1946)
• Substances containing vinyl group or their amino or
alkylamino derivatives are oxidized by periodic acid
to form dialdehydes.
• This combines with Schiff’s reagent to form an
insoluble magenta colored compound.
Result:
PAS positive substances ----- magenta
Nuclei ------------------------- blue
Other tissues ----------------- yellow 13

14. PAS POSITIVE SUBSTANCES
• ADRENAL LIPOFUSCHIN
• AMYLOID
• ACTINOMYCES
• B CELLS OF PITUITARY
• CARTILAGE MATRIX
• CEREBROSIDES
• CELLULOSE
• COLLAGEN
• COMPOUND LIPIDS
• COLLOID FROM THYROID
• GLYCOGEN
• HYALINE CAST (RENAL
TUBULES)
• KERASIN (GAUCHER’S)
• BM
• MEGAKARYOCYTE
GRANULES
• MUCINS
• OVARIAN FOLLICLES &
CYSTS
• PHOSPHOLIPIDS
• RUSSEL BODIES
• STARCH
• OCULAR LENS CAPSULE
• CORPORA AMYLACEA
• ZYMOGEN GRANULES
(PANCREAS)
• RBC
14

15. 15

16. 16

17. PAS+ DIASTASE SENSITIVE MATERIAL IN MCARDLE’S
DISEASE
17

18. 18

19. ALCIAN BLUE
PRINCIPLE:
• Cationic dye forms electro-static bonds with certain
tissue with poly-anions bearing either carboxyl or
sulphate groups.
• Specific.
• Strong colour.
• Insoluble.
• Permanent.
19

20. METHOD:
• Dewax sections & wash with water.
• Alcian blue - 5mins & wash with water.
• Counterstain with 0.5% aqueous neutral red - 2-3
mins.
• Wash in water, rinse in alcohol.
• Clear and mount.
ALCIAN BLUE
20

21. RESULTS:
• Acid mucin ----- blue
• Nuclei ---------- red
• Alcian blue solutions of varying pH is used to
separate and identify the different acid mucins.
ALCIAN BLUE
21

22. 22

23. 23

24. 24

25. COMBINED ALCIAN BLUE – PAS
Principle:
• Staining first with Alcian blue will stain all acid mucins including
those that are PAS +ve so that they don’t react with PAS only
neutral mucins.
Results:
Acid mucins : blue
Neutral mucins : magenta
Mixture : colour will depend on dominant entity Blue-purple-violet
or mauve
Note: Stain lightly with haematoxylin.
25

26. 26

27. ALCIAN BLUE
The pH of this stain can be adjusted to give more specificity.
Alcian blue stains are simple, but have a lot of background
staining.
PAS
Stains glycogen as well as mucins, but tissue can be pre-
digested with diastase to remove glycogen.
The stain that is the most sensitive is PAS, but we must learn
how to interpret it in order to gain specificity.
MUCICARMINE
Very specific for epithelial mucins.
27

28. STAINS FOR GLYCOGEN
28

29. BEST’S CARMINE STAIN
PRINCIPLE:
• Staining is by hydrogen bond formation between
OH groups on the glycogen and hydrogen atoms
of the carminic acid.
• Method is highly selective rather than specific, as
fibrin and neutral mucin also stain weekly.
RESULTS:
Glycogen ---------------- deep red
Some mucin, fibrin ------ weak red
Nuclei--------------------- blue
29

30. 30

31. 31

32. 32

33. DEMONSTRATION BY ENZYME METHOD
• Glycogen is destroyed by diastase obtained
by saliva and malt.
• Diastase reacts with glycogen present in the
section and removes it and subsequent
staining of this section shows loss of staining
when compared to untreated section.
33

34. STAINS FOR LIPIDS
34

35. IDENTIFICATION OF LIPIDS
• Solubility
• Lipids - colorless
• Examination by polarized light (Isotropic, anisotropic)
• Reduction of osmium tetra oxide
• Demonstration by fat soluble dyes
• Other methods
35

36. DEMONSTRATION WITH FAT SOLUBLE
DYES
• Based on the fact that the dyes are more soluble in fat than in
the solvent employed.
• Sudan III (Daddi 1896)
• Oil red O in tri-ethyl phosphate
Oil red O Sudan Black B
Fat -----brilliant red
Nuclei---blue
Unsaturated ester & triglycerides
-----------------blueblack
36

37. LIPIDS
Fats Oil red O, Sudan black
Lipofuschin Sudan black, Autofluroscence
Phospholipids Ferric Haematoxyline
Sphingomyelin Ferric Haematoxyline
Gangliosides PAS
Cerebrosides PAS
Cholesterol (free) Filipin
37

38. 38

39. 39

40. OSMIUM TETROXIDE PARAFFIN
PROCEDURE
• PRINCIPLE: osmium tetroxide chemically binds with
the fat to give black colour.
• This is the only method for demonstration of fat on
paraffin sections.
• Fat ------------------------ Black
• Other tissue elements --- According to method used.
40

41. 41

42. STAINS FOR
COLLAGEN
42

43. MASSON’S TRICHROME STAIN
• Solution A – Acid fuchsin, Glacial acetic acid, Distilled water
• Solution B – Phosphomolybdic acid, Distilled water
• Solution C – Methyl blue or light green, Glacial acetic acid,
Distilled water
RESULTS:
Nuclei-------------------------- blueblack
Cytoplasm, Muscle, RBC ---- red
Collagen ---------------------- blue/green 43

44. CHRONIC ACTIVE HEPATITIS WITH
COLLAPSE IN LIVER, TRICHROME
STAIN.
44

45. MASSON’S
TRICHROME STAIN OF
SKIN
45

46. VAN GIESON TECHNIQUE
(1899)
• Saturated aqueous picric acid solution (acidic PH)
• 1% aqueous acid fuchsin solution(stain the collagen)
• Distilled
RESULTS:
• Nuclei --------------------------------blue/black
• Collagen ---------------------------- red
• Other tissues ----------------------- yellow
46

47. VAN GIESON STAIN
47

48. USES
1. For showing ragged red fibres in mitochondria myopathy.
2. For inclusion in rod myopathy – dark red to purple
3. In the evaluation of the type and amount of extracellular
material.
4. Muscular dystrophies.
5. Differentiating muscle from connective tissue.
6. Demonstration of fibrin or young collagen fibres.
48

49. ELASTIC FIBERS
• Verhoeff’s iron hematoxylin as providing the greatest
contrast and orcein is the simplest.
Verhoeff’s iron hematoxylin
(hematoxylin-ferric chloride - iodine
solution )
Orcein
Elastic ---------------- black
Collagen-------------- red
Muscle --------------- yellow
Nuclei ----------------- black
Elastic ------dark brown
 Copper associated protein,
elastic fibers,
hepatitis B surface antigen: dark
brown
49

50. Orcein stain
Verhoeff’s
iron
hematoxylin
stain
50

51. MOVAT’S PENTACHROME
STAIN
• Demonstration of mucin, fibrin, elastic fibers, muscle,
and collagen.
• Five different stains.
• Acidic mucosubstances are stained by alcian blue.
• Iron hematoxylin will stain the elastic fibers
• Crocein scarlet and acid fuchsin will stain the muscle,
cell cytoplasm, collagen and ground substances.
51

52. • NUCLEI AND ELASTIC FIBERS.....................…..…...BLACK
• COLLAGEN.......................................................YELLOW
• GROUND SUBSTANCES AND MUCIN.......................BLUE
• FIBRINOID, FIBRIN....................................INTENSE RED
• MUSCLE............................................................RED
52

53. 53

54. GORDON & SWEET’S METHOD FOR
RETICULIN FIBERS
• 10 ml of 10 % KOH
• 40 ml of 10 % Silver Nitrate
RESULTS:
Reticulin fiber ----- black
Nuclei ------------- gray
Background ------ red
Other tissues ----- according to counter stain
Appearance of reticulin pattern particularly useful in
the following situation.
1. Liver biopsies : bridging nacrosis and massive hepatic
necrosis.
2. Lymph reticular neoplasms
54

55. 55

56. FIBRIN
• An insoluble fibrillar protein – formed by polymerization of
fibrinogen.
• Commonly seen areas of tissue damage.
RESULTS:
1. H & E ------------------------ pink
2. PTAH ------------------------ blue
3. Masson’s trichrome--------- red
4. MSB-------------------------- red 56

57. PTAH(PHOSPHOTUNGSTIC ACID
HEMATOXYLIN)
• Phosphotungstic acid
• Hematoxylin
• Distilled water
• For the demonstration of cross-striations in skeletal muscles
and fibrin.
RESULTS:
Fibrin, neuroglial fibers ------- blue
Bone, cartilage matrix --------- shades of yellow
Reticulin, elastin ---------------- orange to brownish red
57

58. 58

59. 59

60. MSB (MARTIUS SCARLET & BLUE
STAIN)
• Martius yellow
• Brilliant crystal scarlet
• Phosphotungstic acid
• Methyl blue
• Glacial acetic acid
RESULTS:
Nuclei -------------------------------- blue
RBC ---------------------------------- yellow
Muscle ------------------------------- red
Fibrin -------------------------------- red
Collagen ---------------------------- yellow
60

61. MSB STAIN
61

62. Stains
Tissue
V.G. MT PTAH PAS RETIC H & E
Collagen
Red Blue
Green
Orange-
red
+ Grey Deep pink
BM Yellow Blue
Green
Orange
+++
+
Grey pink
Reticulin Yellow Blue
Green
Orange-
brown
++
Blac
k
-
Elastic Yellow Orange-
brown
- Pink
Fibrin Yellow Red
Blue
+- Gray Pink
Verho
eff
Red
-
-
BLAC
K
-
62

63. AMYLOID
• It is considerably easier to demonstrate the amyloid on
fresh frozen section than in paraffin section.
• On fresh tissue by treatment with iodine and potassium
iodide. The deposit is stained a nutmeg-brown color, which
changes to blue-violet by treatment with dilute sulphuric
acid.
63

64. H & E Amorphous Pink Missed if small
deposits
Methyl/crystal
violet
Metachromatic
pink
Technically
unsatisfactory.
PAS Pale pink or
negative
No diagnostic
use
Van Gieson
Khaki
Not specific
Pepsin digestion Pale pink if eosin
used
Specific but
difficult.
Congo red
Orange-red Best.
64

65. 65

66. STAINS FOR NUCLEIC
ACID
66

67. FEULGEN NUCLEAR REACTION
PRINCIPLE:
• Mild acid hydrolysis using 1M HCl at 60ºC breaks the
purine deoxyribose bond, the resulting ‘exposed’
aldehydes are then demonstrated by Schiff’s reagent.
• Ribose purine bond is unaffected because glycol group of
ribose is substituted by phosphate
RESULTS:
DNA ------------- red purple
Cytoplasm ------ green.
67

68. FEULGEN REACTION
68

69. PIGMENTS
• ARTIFACTUAL: Formalin, Mercury, Dichromate
• EXOGENOUS: Carbon, Silica, Asbestos
• ENDOGENOUS:
1. Haematogeous: Hemosiderin, Hemoglobin, Bile
pigment
2. Non-haematogenous: Melanin, Lipofuscin,
Chromaffin
3. Minerals: Calcium, Copper, Urates
69

70. PERL’S PRUSSIAN BLUE FOR IRON
• Haemosiderin contains iron in the form of ferric hydroxide.
Perl’s reaction
Haemosiderin + HCl Fe+3
ferric ferro cyanide Pottasium Ferro
cyanide
RESULTS:
Ferric iron ------------------------- dark blue
Tissue nuclei ----------------------- red
70

71. 71

72. 72

73. HEMOGLOBIN
• The enzyme hemoglobin peroxidase can be demonstrated
by benzididne-nitropruisside method & patent blue method.
• Leuco patent blue V method
• 1% aqueous patent blue V
• Powdered zinc
• Glacial acetic acid
RESULTS:
• Hemoglobin peroxidase ------ emerald to blue green
• Muscle -------------------------- yellow
• Collagen ------------------------ red
73

74. STAINS FOR BILE PIGMENTS
MODIFIED FOUCHET’S TECHNIQUE
PRINCIPLE:
Bile pigment is converted to green colour of biliverdin
and blue cholecyanin by oxidative action of ferric
chloride in presence of trichloracetic acid.
RESULTS:
• Bile pigments ------------ emerald to blue green
• Muscle ------------------- yellow
• Collagen ---------------- red
74

75. BILE PIGMENT IN H&E
STAIN
75

76. ENDOGENOUS
PIGMENTS: BILE
PIGMENTS
BILE PIGMENT –
FOUCHET’S METHOD
76

77. MASSON-FONTANA METHOD
• Silver solution - 10 % silver nitrate
- Ammonia
RESULTS:
• Melanin, argentaffin, lipofuscin ---- black
• Nuclei --------------------------------- red
77

78. Fontana masson silver stain for melanin 78

79. 79

80. STAINS FOR LIPOFUSCINS
• Periodic Acid Schiff’s Method.
• Schmorl’s Reaction.
• Masson Fontana Silver Method.
• Basophilia-using Methyl Green.
• Long Ziehl-neelsen Method.
RESULTS:
• Lipofuscin ---------- Magenta
• Nuclei -------------- Blue
• Background ------- Pale Magenta to pale Blue.
80

81. 81

82. LIPOFUSCIN
PIGMRNT BY
MASSON FONTANA
STAIN
82

83. STAINS FOR LIPOFUSCINS
Sudan black B method.
• Results: Lipofuscin & RBC - black.
Background - pale grey.
Gomori’s aldehyde fuscin technique.
• Results: Lipofuscin - purple
Background - yellow.
83

84. STAINS FOR MINERALS
Copper:
1. Rubeanic Acid Method:
Results: Cu- Greenish Black.
Nuclei- pale Red.
2. Modified Rhodanine Technique:
Results: Cu- Red To Orange Red.
Nuclei- blue.
Bile- Green.
84

85. 85

86. STAINS FOR CALCIUM
1. H & E: Osteoid Tissue ---- Pink
Calcified Bone --- Purplish Blue
Nuclei ------------- Blue
2. Alizarin Red S:
Calcium Deposits ------- Orange-red
3. Modified Von Kossa Method
(Silver Nitrate Substitutes Calcium In The Bone)
Results: Mineralised Bone ---------- Black
Osteoid --------------------- Red 86

87. VON KOSSA'S METHOD
FOR CALCIUM
87

88. 88

89. STAINS FOR URIC ACID &
URATES
• Urates can be extracted by saturated aqueous lithium
carbonate solution.
• Lithium Carbonate Extraction- Hexamine Silver Technique
(Gomori 1936, Grocott 1955)
• Grocott’s Hexamine Silver Solution
• Lithium Carbonate
• Sodium Thiosulphate
• Results :- Light Green
• Methenamine Silver Stains Urates Black.
• Sodium Urate Crystals Are Also Birefringent On Polarization.
• Using A Red Plate, The Crystals Show Negative Birefringence
(Yellow Color)
89

90. H&E STAIN AND
POLARIZED
MICROSCOPY OF
URATE CRYSTALS
90

91. ARTEFACTUAL PIGMENT
Formalin Dark brown/Black
granules
(Non-birefringent)
Alcoholic
picric acid
Malarial
Pigment
Dark brown/Black
granules
(birefringent)
Alcoholic
picric acid
Mercury
Pigment
Coarse black
granules
Iodine,
thiosulphate
Dichromate
Pigment
Fine yellow
deposit
Acid alcohol
91

92. FORMALIN
PIGMENT IN
LYMPHOID TISSUE
92

93. BACTERIA (1-14µM):
1. The Gram Stain
2. Ziehl-neelsen (ZN)
3. Gimenez (For H. Pylori)
4. Levaditi’s Method
FUNGI (2-200µM):
1. Grocott (Hexamine) SM
VIRUSES (20-300µM):
1. Orcein For Hepatitis B Surface Ag
PARASITES:
1. PAS
STAINS FOR ORGANISM
93

94. STAINING OF FUNGI
• H&E: Practically all fungi but in many cases contrast in
tissue not great.
• Gram’s stain: Most fungi, central bodies of capsulated
fungi are strongly positive. Eg: Cryptococcus, blastomyces
• Giemsa stain: Some species of nocardia. Eg: N.
Asteroids, Braciliensis, Club end of Actinomyces.
• Metachromatic stain: Toludine blue. Eg: Bodies of
Cryptococci, Blastomycosis
• Negative stain: India ink preparation. Eg: Capsule of
Cryptococci. 94

95. Mucicarmine ---------- Cryptococci
PAS -------------------- Fungal Capsule
• Gomori’s Silver Methanamine (SM)
Fungal Wall Components + Chromic Acid
Oxidation
Aldehyde Group
AgNO3 Black Metallic Silver
Reduction
Results :- Fungi ------------------ Black
Background----------- Pale Green
RBC ------------------- Yellow
95

96. 96

97. Staining Methods Application
H & E Morphology
PAS BM, Mesangium
PTAH Fibrin
Congo red Amyloid
Verhoeff’s Elastic fibers
STAINING METHODS FOR RENAL
BIOPSIES.
97

98. Staining Method Application
PAS Glycogen & other
Perls’ Iron
Long ZN Lipofuscin
SBB Fat, Lipofuscin
Reticulin Reticulin network
PTAH Fibrin
Congo red Amyloid
STAINING METHODS FOR LIVER
BIOPSIES
98

99. 99

100. 100

101. RECENT ADVANCES
101

102. 102

103. REFERENCES
1. Cellular Pathology Technique - C.F.A Culling, R.T.Allison
2. Theory And Practice Of Histological Techniques -
Bancroft
3. Laboratory Manual Of The Armed Forces
4. Histopathological Technique - Lynch
5. Lab Techniques In Surgical Pathology - Shameem
Shareef
103