Dr. Ajit Surya Singh

1. Basic Histopathological
Techniques
Dr . Ajit Kumar Singh
1st year PGT MD (Laboratory medicine)
Chittaranjan National Cancer Institute

2. PROTOCOLS FOLLOWED IN HISTOTECHNIQUES
1. Receipt & Identification
2. Labeling of the specimen with numbering
3. Fixation
4. Grossing
5. Dehydration
6. Clearing
7. Infiltration
8. Embedding
9. Section cutting
10. Staining
11. Mounting

3. Receipt & Identification,
Labeling of the specimen with
numbering
• Patient information
• Clinical history
• Site of origin of specimen
• Lab number (unique identification)

4. Fixation
• Chemical process by which biological tissues
are prevented from decay (autolysis or
putrefaction)
1. Preserves original shape, size & structure.
2. Prevents autolysis (acts against hydrolytic
enzymes)
3. Stops the bacterial effect.

5. TIME OF FIXATION :
Medawar (1941)
t=(d/k)^2
d is depth reached by fixative
k is Coefficient of Diffusability
.79 10% formaldehyde
1.0 100% ethanol
1 hour – 0.8 mm penetration.
2 hours – 1.2 mm penetration.
4 hours – 1.6 mm penetration
25 hours – total 1 cm thick block (5 mm from each side)
d= k√t

6. CHOICE OF FIXATIVE :
Routine 10% buffered Formalin
Bone marrow Zenker’s formal (Helly’s fluid)
biopsies
GI biopsies Bouin’s fluid (Picric acid)
Bone Formic Acid in 10% HCl
Adrenal medulla Hollande’s
Brain tissue Formalin ammonium bromide
E/M specimen Gluteraldehyde ( preffered)
Zamboni’s
Flemming’s
Schaudinn’s
10 % formol alcohol

7. CHEMICAL FIXATIVES
COAGULANT FIXATIVES
ALCOHOL (PRECIPITANTS)
ETHANOL, METHANOL
TRICHLOROACETIC ACID
ACETONE
PICRIC ACID
ACETIC ACID
NON COAGULANT
(CROSS LINKING )
FORMALDEHYDE
GLUTERALDEHYDE
OSMIUM TETROXIDE
POTASSIUM DICHROMATE
MERCURIC CHLORIDE
ZINC CHLORIDE
CHROMIUM TETROXIDE

8. FORMALDEHYDE
MOA :
In aqueous solution formaldehyde forms methylene hydrate
Reacts with several side chains of proteins eg. Lysine, cysteine ,
histidine, arginine, tyrosine, and reactive hydroxyl group of serine and
threonine

9. Penetrates tissue rapidly
Slight shrinkage in tissue
Preserves all structural details
Reacts with phosphatidyl ethanolamine causing degradation but not
with other lipids like cholesterol , cerebrosides , sulphatides and
sphingomyelins
Loss of carbohydrate is high yet it can be demonstrated satisfactorily.

10. • The cross linkage does not harm protein
structure, so antigenicity is not lost, hence
Formalin is suitable for immunohistochemistry.

11. • End point- change in colour of blood can be
accepted as an end-point of formalin fixation
FORMATION OF ACID FORMALIN HEMATIN
MAKES BLOOD TURN TAN BROWN

12. GROSSING
5.Assess margins
4.sample
3.ink
2.measure
1.orient

14. Tissue Processing
• In order to cut thin sections of the tissues, it
should have suitable hardness and consistency
when presented to the microtome.

15. Tissue Processing
• Dehydration
• Clearing
• Infiltration
• Embedding

16. Dehydration
 It is removal of ‘free’ unbound water and aqueous fixatives from the
tissue components.
 Done by passing the tissue through increasing concentrations of
dehydrating agents. (GRADED DEHYDRATION)
 If concentration gradient is excessive/ drastic, diffusion currents
across the cell membranes increase the possibility of cell distortion.
 Excessive dehydration -- tissue become hard, brittle and shrunken.
 Incomplete dehydration -- impair the penetration of the clearing
reagents
-- tissue remains soft and nonreceptive to
infiltration.

17. Tissues are dehydrated by using increasing strength of alcohol;
i.e. 70%, 90% and 100%.
The duration for which tissues are kept in each strength of
alcohol depends upon the size of tissue, fixative used and type of
tissue.
Delicate tissue will get high degree of shrinkage by two great
concentration of alcohol. ( start with less concentrations)
The volume of alcohol should be 50-100 times that of tissue.

18. In automated processors–
 Anhydrous CuSO4 can be added to last jar of dehydrating
agent.
 Indicates completion of dehydration.

19. Clearing
 Clearing reagents act as an intermediary b/w the dehydration
and infiltration solutions.
 They should be miscible with both solutions.
 As paraffin wax is not alcohol soluble, we replace alcohol with a
substance in which wax is soluble.
 When the dehydrating agent has been entirely replaced by most
of these solvents the tissue has a translucent appearance: hence
the term ‘clearing agent’.

20.  Most clearing agents are aromatic hydrocarbons or short-
chain aliphatic hydrocarbons.
 Most clearing agents are flammable liquids, which warrant
caution in their use
 Clearing agents with a low boiling point are generally more
readily replaced by paraffin wax.

21. Criteria for suitable clearing agent:
 Rapid removal of dehydrating agent
 Ease of removal of melted paraffin
 Minimal tissue damage
 Low toxicity
 Cost

22. CLEARING AGENTS
 Xylene- Routine
Recyclable
It is suitable for clearing blocks that are <5 mm in
thickness
Overexposure -- hardening of tissues.
 Toluene- flammable and volatile
 Chloroform- toxic
 Methyl benzoate and methyl salicylate
 Citrus fruit oils- pungent odour

23.  In infiltration, tissue is impregnated with a
medium, forming a matrix within the cellular
spaces and preventing distortion of the tissue
structure during microtomy.
 Embedding is enclosing of properly processed
correctly oriented specimens in a medium
that provides external support.
INFILTRATION AND EMBEDDING

24. Tissue that come off the tissue processor are still
in the cassettes and must be manually put
into blocks by technician who must pick the
tissues out of the cassette and pour molten
paraffin over them.
This embedding is very important because the
tissues must be aligned or oriented properly in
the block of paraffin.
INFILTRATION AND EMBEDDING

25.  Commercial embedding centres, which combine a heated mould store,
a molten wax reservoir/dispenser and a cold plate, are readily available
 Paraffin wax is dispensed automatically from a nozzle into a suitably
sized mold. The tissue is oriented in the mold; a cassette is attached,
producing a flat block face with parallel sides
 Once the wax has solidified, the tissue blocks may be gently removed
from their moulds and prepared for microtomy.

26. Reagent minutes
• 70% Formol alcohol 60
• 80% Alcohol 60
• 90% Alcohol 60
• 100% Alcohol 60
• 100% Alcohol 60
• Copper Alcohol 60
• Copper Alcohol 60
• Aniline 360
• Xylene 60
• Xylene 60
• Paraffin wax 90
• Paraffin wax 90

27. Microtomy
 Process in which tissue is sectioned and attached to a surface for
further microscopic examination.
 The basic instrument used in microtomy is the microtome
 Advancing mechanism moves the object (paraffin block) for a
predetermined distance until it is in contact with the cutting tool
(knife or blade). The specimen moves vertically past the cutting
surface and a tissue section is produced.
 Types of microtome used: Rotary
Base sledge
Rotary rocking
Sliding
Ultramicrotomes

28. PARAFFIN SECTION CUTTING
• Equipments required:
 Floatation Bath
 Slide drying oven or hot plate
 Fine pointed or curved forceps
 Sable or camel haired brush
 Scalpel
 Slide rack
 Clean slides
 Teasing needle
 Ice tray
 Chemical resistant pencil or pen

29. Floatation bath:
Used for floating out tissue ribbons after sectioning
Temperature in the bath should be 10°C below the melting
point of the paraffin to be sectioned
Prevent water bubbles from being trapped under the section (
use distilled water)
Add alcohol/ drop of detergent- ↓surface tension- allowing
section to flatten out with ease

30. Brush and Forceps:
Removal of folds, creases and bubbles formed during
floatation of section on water bath.
Manipulating the section as it passes across the edge of the
blade.

31. Drying oven/ Hot plate:
Keeps warm air circulating around the slides
Temp should be ~ melting point of paraffin
Special care- CNS (↓ T to prevent splitting and cracking of
tissues)

32. Slides:
75mm*25 mm; 1-1.2 mm thick
Larger slides for brain and eyes
Positively charge or pre treated with an adhesive resist
detachment of the tissue from the slide during staining

33. Protein adhesives-
 albumin, gelatin, starch
 Prone to bacterial overgrowth and heavy staining
Poly L Lysine :
 0.1% solution; further diluted for use, 1 in 10 with distilled water
 Diminish with time
APES (3 –Aminopropyltriethoxysilane):
 cytology, esp specimens that may be bloody or contain proteinacious material

34. Charged or plus slides:
Manufactured with permanent positive charge
Coating the slides with a basic polymer in which a chemical
reaction occurs leaving the amino groups linked by covalent
bonds to the silicon atoms of the glass.
Superior in their resistance to cell and tissue loss during
staining or pre treatment such as enzyme and antigen
retrieval.

35. Cutting section
 Trimming of tissue blocks
 Blocks are arranged on a cooling device, to cool both tissue and the
paraffin.
 A small amount of water is adsorbed into the tissue causing slight
swelling, making sectioning easier.
 Oversoaking may cause expansion and distortion of the tissue section.
 Proper processing eliminates the need to pre soak blocks.
 3-4 microns
 Smooth slow stroke

36. Floating out sections
 Must be smooth with the trailing end of the ribbon making
contact with the water first.
 Slight drag produced when the rest of the ribbon is laid on the
water is sufficient to remove any folds that occur.
 Teasing with forceps
 30 seconds- time for a ribbon to flatten; prolonged time causes
excessive expansion, distorting the tissue

37. Drying sections:
 Temperature should be at the melting point of paraffin
 It is important to eliminate overheating during the slide
drying stage, as cellular details may be compromised.
 For delicate tissues reduce temperature for prolonged time

38. Cutting hard tissues:
The reason for cutting difficulties is more likely poor fixation or
over-processing.
Prolonged soaking of the block
Exposing the block surface to running tap water for 30 minutes
Slight reduction in knife slant

39. Decalcification
• Calcium deposits are hard and cannot be cut by
microtome.
• Calcium is removed prior to embedding to allow
sectioning.
• This is done by HNO3, HCl - strong and rapid action (on
cortical bone), but also damage cell morphology. Not
done for bone marrow.
• Acetic acid and Formic acid are used for bone marrow
but are slow acting
• Formic acid in 10% HCl concentration all rounder
• EDTA is also used for decal

40. Staining
• Hematoxylin (basic dye) and
• Eosin (acidic dye)

41. Hematoxylin
• Hematoxylin is the most widely used stain
• Hematoxylin is extracted from the heartwood [logwood] of
the tree Hematoxylon campechianum, with hot water &
then precipitated out from the aq. sol. of urea
• Was originated Mexican state of Campeche
• But is now cultivated in the West Indies

42. Hematoxylin
• Haematoxylin by itself does not have staining property
• Oxidation product “ haematin”- the actual staining compound
• Synthetic dyes have been recommended as replacements
 Celestine blue (CI 51050)
 Gallocyanine (CI 51030)
 Gallein (CI 45445)
 Erichrome cyanine R (also called chromoxane cyanine R)
 Solochrome cyanine (CI 43820).

43. • The process of oxidizing haematoxylin to haematin is called
RIPENING
Methods of ripening
Natural oxidation Chemical oxidation
By exposure to natural light and air
By adding sodium iodide (Mayer’s
haematoxylin) or mercuric oxide
(Harris’s haematoxylin)
Slow process ( 6 to 8 weeks) Ready to use instantly
Retains stain for very long time since it
is completely oxidized.
Short lived staining since the
continuing oxidation destroys much of
haematin to a colourless component.
Eg: Ehrlich’s haematoxylin
Delafield’S haematoxylin
Eg: Mayer’s haematoxylin
Harris’s haematoxylin

44. Mordant
• Haematin is anionic and has a poor affinity for tissues. So, nuclear
staining needs a MORDANT to be added
• The mordant /metal cation confers a net positive charge to the
dye mordant complex & enables it to bind to anionic tissue
sites such as nuclear chromatin
• The cations used as mordants are:
 Aluminium
 Iron
 Tungsten
 Lead ( argyrophyl cells)
 Molybdenum

45. Blueing
• Nucleus is stained red colour by all stain which is converted to blue-
black when the section is washed in a weak alkali solution
• Bluing agents:
 Running tap water
 Hot water
 2% NaHCo3
 1% LiCo3
 Scott’s tap water
 Marble chips
 1% ammonia vapor
 Aluminium solutions

46. Differentiation
• Removing the dye molecules from
cytoplasm while keeping the nucleic acid
complex intact
• 1% Hcl

47. Eosin
 Most suitable stain to combine with an alum hematoxylin
 It has good ability to distinguish b/w the cytoplasm of different
types of cells & b/w different types of connective tissue fibers
& matrices by staining them different shades of red & pink
 Eosins are xanthene dyes
 Commercially available types are
---Eosin Y (Eosin yellow ,eosin water soluble)
---Ethyl eosin ( Eosin S, alcohol soluble-not used )
---Eosin B (Eosin bluish- used in hematology )

48.  Eosin Y is the most widely used
**Both water & alcohol soluble
**Used as 0.5% or 1% solution in distilled water
**A crystal of thymol is added to inhibit fungi
**Acetic acid is added to sharpen the stain
 Eosin B & ethyl eosin- used rarely
 Alternative red dyes of Eosin- Phloxine & Biebrich scarlet

49. • Counterstaining with eosin changes the colour
of haematoxylin alum-stained nuclei from blue
to purplish
• This additive colour change may be due to
attraction of eosin anions to positively charged
amino acid side chains of basic nucleoproteins

50. Staining
1. Deparaffinise the section by Xylene
2. Bring the section to water (HYDRATION) [
90% alc ---50 % alc. -- water ]
3. Remove fixation pigment if any
4. Stain in hematoxylin x 10 min
5. Wash in running water x 5 min

51. Staining
6. Differentiate in 1 % acid – alcohol (1% Hcl in
70% alcohol) x 5-10 sec
7. Blued in tap water x 5min or less
8. Counterstain in 1% Eosin Y x 10 min
9. Wash in running water x 1-5 min
10. Dehydrate through alcohol
90% alcohol –10 sec
Absolute alcohol --10 sec

52. RESULTS :--
NUCLEI-------------------------Blue / Black
CYTOPLASM-----------------Varying shades of pink
MUSCLE FIBRES-----------Deep pink / Red
RED BLOOD CELLS-------RED /Orange

53. Mounting
• Slides cleared with Xylene & mounted in
DPX along with cover slip
D - Distyrene (Polysterene)
P - Plasticizer (Tricresyl phosphate)
X - Xylene

54. Thank you