This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.

1. HISTO -TECHNIQUES

2. OVERVIEW
• Definition
• Specimen Receiving
• Fixation
• Grossing
• Tissue processing
• Embedding
• Microtomy

3. Definitions
• Histology – greek – histos + logia (A.F.J.K. MAYER 1819)
– Study of microscopic anatomy of cells and tissues
• Histopathology – Microscopic study of
diseased tissue
• Histotechnology – processing of tissues in
such a manner as to enable microscopy /
study of the tissue

4. AIM
• Obtaining thin enough sections of the tissue
so as to enable light to pass through it

5. SPECIMEN RECEIVING
• Documented policy defining the minimum
demographic details of the patient
• Spill kit – 5% hypochrorite, gauze piece, yellow
bag, gloves
• Sample transportation – covered containers,
in 10% buffered formalin, chutes
• Unique identification number
• Strict Sample rejection criteria

9. “FIX AT A POINT IN TIME”
• Viable tissue is dynamic
• Process by which constituents of cells are fixed in
a chemical so that they will withstand
subsequent treatment with various reagents with
minimum distortion or decomposition and keep
the tissue in as life like manner as possible
• Ideal fixative does not exist
– Prevent degeneration and autolysis
– Harden to enable cutting
– Not distort the cellular constituents
– Support chemicals used in processing

10. TYPES OF FIXATION
• Immersion
• Perfusion – brain, spinal cord, lungs,
embalming
• Vapour
• Spray
• Microwave fixation/Stabilization
• Freeze drying

11. MICROWAVE PRINCIPLE
• Water molecules are dipolar
• Microwave produces alternating
electromagnetic fields
• Water molecules rotate and this energy is
dissipated as heat
• This heat speeds up the process of fixation,
dehydration, clearing and staining

13. FREEZE DRYING
• Fresh tissue immersed in isopentane cooled by
liquid nitrogen at -160° to -180° - Quenching
• Tissue water removed under vacuum, absorbed
by phosphurous pentoxide – sublimation
• Put in embedding medium
• Little chemical alteration, no loss of glycogen
• Modification of this technique useful in enzyme
histochemistry

14. • Standard histological processing abolishes the activity of most
enzymes
• Freeze drying is the optimal method of tissue preservation
maintaining tissue components in their native state.
Conventionally, however, the freeze dried tissue specimens are
fixed and embedded in wax after freeze drying.
• Glycol methacrylate resin has been widely used as an alternative
embedding medium to wax and the activities of a limited number of
enzymes have been shown in tissue fixed in aldehyde and
embedded in resin. Enzyme histochemistry performed on these
resin sections gives good results.

15. PHYSICAL METHODS CHEMICAL METHODS
Heating
Microwaving
Freeze - drying
Aldehydes – Formal dehyde,
Glutaraldehyde
Alcohol – Methyl alcohol, ethyl alcohol
Methacarn
Glacial acetic acid
Bouin’s fluid
Osmium tetroxide
SIMPLE FIXATIVE COMPOUND FIXATIVE
Formal dehyde
Glutaryl dehyde
Ethyl alcohol
Zenker’s fluid
Bouin’s fluid

16. PRINCIPLE OF ACTION EXAMPLES
Cross link proteins Formal dehyde, glutaraldehyde
Dehydration and hence precipitation of
proteins
Methanol, ethanol, acetone
Changes in pH and hence denaturation of
nucleic acids and salt formation
Acetic acid, trichloroacetic acid, zinc acetate
MICRO ANATOMICAL
FIXATIVE
CYTOLOGICAL FIXATIVE HISTOCHEMICAL
FIXATIVE
NUCLEAR FIXATIVE CYTOPLASMIC
FIXATIVE
Formal – saline
Formal – calcium
Buffered neutral
formalin
Zenker’s fluid
Bouin’s fluid
Carnoy’s fluid
Clarke’s fluid
Fleming’s fluid
Champy’s fluid
Formal saline
Formal calcium
Buffered neutral
formalin
Cold acetone
Acid alcohol

17. Formulae
 Formal Saline
 40% Formaldehyde 100 ml
 NACL 9 gm
 Tap Water 900 ml
 10% Buffered Formalin
 40% Formaldehyde 10 ml
 Sodium Hydrogen phosphate 0.4gm
 Disodium Hydrogen phosphate 0.65gm
 Tap Water 90 ml
Buffered formalin prevents formation of pigment acid formaldehyde
hematin formed from hemoglobin at acidic pH.

18. Formulae
Zenker’s fluid
 Distilled Water 1000 ml
 Hg Cl2 50 gms
 K-Dichromate 25 gms
 NaSO4 10 gms
• It provides excellent fixation of nuclear chromatin, connective
tissue fibres and some cytoplasmic features but does not
preserve delicate cytoplasmic organelles such as
mitochondria.
• Currently out of fashion because of the toxicity.

19. Bouin’s fluid
 Sat aq. picric acid 75 ml
 40% formaldehyde 25 ml
 Glacial Acetic acid 5 ml
• Bouin’s is generally used for testicular biopsy fixation because
it preserves nuclei and chromosomes especially well during
meiosis.

20. Glutaraldehyde
• Used for electron microscopy with osmium
tetroxide
• Glutaraldehyde produces nuclear and
cytoplasmic shrinkage while osmium produces
swelling and balances it
• Formalin is not useful for EM because
methanol which is added to commercial
preparations has denaturing action on tissue
components.

21. • The use of 10% NBF, 10% zinc formalin, or 10%
formal saline is recommended, particularly for
demonstrating kappa light chains on mantle and
follicle centre cells.
• A fixation time of 12 hours in 10% NBF showed
optimum staining of all antigens.
• A highly acidic formalin of pH 3.0 produced the
best immunostaining but in our experience at the
expense of morphology. The use of formalin at
pH 5.0 is therefore recommended giving good
morphology and immunoreactivity.

22. Factors affecting fixation
1. Hydrogen ion Concentration & Buffers
1. Satisfactory fixation occurs at pH between 6 and 8.
2. Buffers like phosphate, bicarbonate, s-collidine and
cacodylate are chosen.
2. Temperature
1. Higher temp causes faster fixation
2. Lower temperature preserves tissue better
3. A tissue of 4mm thickness will be adequately fixed in NBF
10 to 20 times the volume in about 8 hours at room
temperature. Fixation time is shortened by 25-40% if
temperature is raised to 40°c.
4. Faster fixation is obtained through agitation.

23. 3. Penetration of fixatives
– Theorotical rate of penetration is one mm in one hour
4. Osmolality of fixatives
– Slightly hypertonic solution (400-450 mos) ideally.
– Hypertonicity causes cell shrinkage
5. Concentration of the fixative

24. 6. Duration of Fixation ideal is 8 hrs
• over fixation causes hardening of tissue and loss of
antigens
6. Volume change – Tissue fixed in formaldehyde and
embedded in paraffin wax shrinks by 33%

25. Fixation Artefacts
 Changes in volume of tissue
 Formalin pigment
 Diffusion of unfixed material
 Chemical changes

26. Decalcification
• Section from bone /heavy mineralised tissue
difficult to obtain
• Methods
1. Acid decalcification – acq nitric acid, formic acid,
HCl, trichloroacetic acid, Gooding and stewart’s
fluid
• Ideal time is 24-48 hrs
2. Other fluids – Von Ebner’s fluid, Perenyl’s fluid
3. Chelating agents – EDTA solution, ideal agent for
EM because minimum artefacts

27. 4. Ion exchange resins – polystyrene
5. Electrolytic method – Electrolytic solution of
HCl and formic acid used
6. Ultrasonic method
7. Surface decalcification – Achieved by
inverting paraffin block in 5% HCl for one
hour, top 30 micron is decalcified.

28. Factors affecting rate of
decalcification
1. Concentration of active reagent
2. Temperature
3. Agitation
4. Density of bone
5. Thickness of the bit

29. Assesment of decalcification
• Needling, knifing, finger nailing
• X ray examination is the best
• Chemical method using ammonia

30. Trimming
 Requirements
 Adequate light
 Sharp instruments
 Adequate water flow
 Wooden board
 Cassettes
 Labels and lead pencil
 Description
 Sections
 Optimum size 2x2x0.3 cm
 Optimum thickness 3-4 mm
 In case of bony fragment, decalcification
 Cassettes

31. Trimming

33. Tissue Processing

34. Tissue Processing
 Principle :
The aim of tissue processing is to embed the tissue in a
solid medium firm enough to support the tissue and give
it sufficient rigidity to enable thin sections to be cut and
yet soft enough not to damage the knife or tissue.
 Stages
 Dehydration – to remove fixatives and water from tissue.
 Clearing – replacing dehydrating fluid with a fluid that is
totally miscible with dehydrating fluid and embedding
medium
 Impregnation – replacing clearing agent with the
embedding medium
 Embedding

35. Dehydration
 Hydrophilic agent is used which attracts water from
tissue
 Used in increasing strengths
 70% is the first solution
 Anhydrous copper sulfate used in last alcohol as it
absorbs water from alcohol
 Examples
 Ethanol
 Methanol
 Methylated spirit
 Isopropyl alcohol

36. Clearing
 Hydrocarbons which have refractive indices similar
to tissue proteins, end point noted by transparent
appearance of tissues.
 Criteria
 Miscible with both dehydrating and embeding agent
 Speedy removal of dehydrating agents
 Easy removal by paraffin
 Examples
 Xylene
 Toluene
 Chloroform
 Citrus fruit oils
 Cedar wood oil

37. Impregnation
 Paraffin wax - the prefered medium,
Melting point  52-56° C
Plastic point – 10° below melting point
 Paraplast – pellet form, melts rapidly
 Alternate embedding media
 Resins
 Agar
 Gelatin
 Celloidin

38. Automated tissue processing
Processing schedule
Container Fluid Time (hrs)
1 10% Formulin 0
2 70% Alcohol 1/2
3 95% Alcohol 1/2
4 100% Alcohol 1/2
5 100% Alcohol 1
6 100% Alcohol 1
7 100% Alcohol 1
8 100% Alcohol/Xylene 1/2
9 Xylene 1
10 Xylene 2
11 Wax 21/2
12 Wax 4

39. Important points
Bone, skin and CNS tissues require three
changes of wax
Two changes of wax suffice for other organs

40. Embedding
 Requirements
 Embedding media
 Cold plate / ice plate
 Wax Dispenser
 L-molds / ice trays / paper blocks / tissue tek system
 Wooden blocks
 Forceps
 Spatula
 Typed labels

41. Embedding

42. Embedding

44. Embedding media
 Water insoluble media
– Paraffin wax
– Carbowax
– Plastic embedding medium – Butyl methacrylate
– Acrylic
– Epoxy resin
– Epon
– Maraglas 655
– Polyester resin
Water soluble media
– Durcupan
– Aquon
– Glycol methacrylate

45. Important points
• Size of the mould should be such that there is a
margin of 1-2 mm around the tissue
• Embed all bits of skin / cervical cone only few
mm apart in the same block with parallel
epithelial edges
• Endometrial samples and cell blocks are best
kept at centre of block and tamped to the
bottom
• Stand mount – cyst walls, gall bladder walls,
ovarian wedges and skin sections – required edge
kept at the bottom

46. • Donot embed small and large specimen in the
same block, smaller may be lost in obtaining a
better section of the larger one
• Muscle biopsies sectioned both longitudinally
and transversely
• Tubular structures are cut transversely

47. Paraffin wax additives
• Increase hardness or alter structure to give
proper sectioning
• Examples
– Ceresin
– Bees wax
– Bay berry wax
– Micro crystalline waxes

48. Special embedding techniques
• Double embedding
– Impregnated in celloidin, blocked in paraffin wax
– Used in making blocks of tissues with varying
consistency such as eyes where retina is easily
detached
• Tissue mat and Bioloid
– Thin walled and circular sections maintain their
original shape due to elasticity of the media

51. Paraffin section cutting
 Requirements:
 Microtomes
 Disposable blades
 Water bath
 Fine forceps
 Small squirrel hair brush
 Slide rack
 Clean slides
 Ice tray
 Diamond pencil

52. Microtomes
 Precision instruments that cut sections from the
paraffin blocks, thin enough for examination
under microscope.

53. Sliding microtome
• Block is stationary, knife moves horizontally
against it

54. Rocking microtome
• The knife is fixed, blok of tissue moves
through an arc and strikes against the knife

55. Sledge Microtome
• Similar in function to sliding microtome

56. Rotatoty Microtome

57. Auto cut microtome

58. Vibrating knife microtome
• Sections obtained without fixation /impregnation /freezing,
useful for histochemistry
• Sections are thick and not suitable for routine staining

59. Ultracut microtome
• Used for cutting 500 to 600 A° thin sections for EM

60. MICROTOME KNIFE
• Vicker’s Hardness number
– Important to use knives of gauranteed hardness
– VHN = 1.72p the desirable hardness is 400-900 VHS
D²
Where D is the diagonal between corners of rectangular indentation, P is standard load

61. one side is flat, other is concave
used for cutting nitrocellulose blocks
originally designed for frozen sections,
now used for all microtomes
Used to section hard tissues such as
undecalcified bone

62. Knife materials
Knife material Use
Steel / Disposable knives
Sintered tungsten carbide on
steel
Diamond knives
Glass knives
Routine
Undecalcified bone and acrylic
resin
EM for sectioning epoxy resin
blocks
Ultramicrotomy

63. Sharpening of knives
• Manual
– Honing
• Coarse
• Fine
– Stropping
• Automatic

64. Honing
• Honing is removing nicks and irregularities
from the knife edge
• Hones are sharpening stones
– Examples – Belgian Vein Black and Arkansas
• Lubricants used during the process to act as
coolants and flow away fine particles

65. Honing
• Abrasive powders
– Diamond
– Carbodundum (silicon carbide)
– Aluminium oxide
– Iron oxide
– Ceric oxide
– Chromium oxide

66. To watch stropping and honing, search for the same on you tube… many videos are available

67. Stropping
• Polishing a fairly sharp knife
• Strops are made of hide from the rump of
horse

68. Knife Sharpening machine

69. Fine Cutting

70. Sections

71. Section adhesion
• Adhesives used
– Poly L – lysine
– APES
– Mayer’s Egg Albumin
– Gelatin
– Starch
– Resin
– Sodium silicate
– Celloidin

72. Difficulties commonly encountered in
sectioning
 Failure of block to ribbon
 Too hard paraffin
 Knife
 Uneven Ribbons
 Irregular knife edge
 Knife not parallel to the block
 Impure paraffin
 Alternate thick and thin Ribbons
 Too soft wax
 Loose block or blade
 Faulty microtome

73. Difficulties commonly encountered in
sectioning (Contd.)
 Tight coil of sections
 Blunt blade
 Too thick sections
 Adherence of sections to knife
 Dirty knife edge
 Dull knife

74. Frozen sections & Cryostat
Intorduced by Louis B. Wilson in 1905
Cryostat is a special microtome refrigerated to
-20°C, tissue placed in it has water which is
converted to ice, thus making the tissue hard
and providing a self embedding medium
Applications:
 Rapid diagnosis
 Assessing margins
 Confirm / refute metastasis
 Demonstration of fat which is lost during routine
processing

75. Cryostat

76. Cryostat

77. Cryostat