A tissue processor is used to prepare tissue samples for analysis by fixing, staining, dehydrating or decalcifying them. The techniques for processing the tissue, whether biopsies, larger specimen removed at surgery

1. Tissue Processing

2. Introduction
 Fresh Tissues are soft hence cannot be cut on
microtome.
 In order to get thin sections of good quality
for final diagnosis , the tissue needs firm
solid medium which gives sufficient rigidity
to ensure thin sectioning.
 Because water & wax are not miscible,
tissues must be gradually dehydrated with
alcohol.

3. Important maintenance tips
 • Any spillage or overflow should be cleaned immediately.
 • Accumulation of wax on any surface should be removed.
 • The temperature of the paraffin wax bath should be set
to 3°C above the melting point of the paraffin wax and
monitored daily
 • Timings should be checked when placing tissue cassettes
in the processor, especially when delayed schedules are
selected
 • Warm water flushes should be incorporated, keeping the
lines free of salts, protein and debris

4. Principle
 The Principle of tissue processing is to remove all
extractable water from the tissue replacing it with a
support medium that provides sufficient rigidity to enable
sectioning of the tissue without damage or distortion.
The steps involved are:-
1) Dehydration :- removal of water & fixative from the
tissue.
2) Clearing:- Removal of dehydrating solution , thus making
the tissue components receptive to infiltration medium.
3) Impregnation:- removal of clearing agent & infiltration of
paraffin wax in order to give internal support to tissue.

8. Dehydration
 Removes free or unbound water molecule of the tissue as
the supporting medium (paraffin) is not miscible with
water.
 • Sharp difference of concentration gradient of the
dehydrating fluid may damage the delicate tissue.
 Gradual dehydration is necessary.
 Too much time in the dehydrating fluid: the tissue
becomes hard and brittle.
 • Routine laboratory: 70, 90 and 100% alcohol for 2 h
each.

9.  Common dehydrating agents:
 – Ethyl alcohol
 – Methylated spirit
 – Methanol
 – Butyl alcohol
 – Isopropyl alcohol
 – Dehydrating agents other than alcohol:
dioxane, ethylene glycol and acetone

10. Comparison of different dehydrating agents
Dehydrating
agents
Advantages Disadvantages
Ethyl alcohol • Rapid and efficient dehydrating
agent
• Needs licence from the government
• Inflammable
• Hard and brittle tissue if kept for long
time
Methanol Equally effective as ethanol • Volatile
• High cost
Isopropyl
alcohol
• Relatively rapid action
• Non-toxic
• Minimal tissue shrinkage
• Not possible to use in celloidin
technique
Dioxane • Rapid action
• No shrinkage of tissue
• Highly toxic gas is generated
Ethylene glycol Rapid
• No graded solution is needed
• Tissue can be kept in it for long time
Very expensive
• Clearing agent is needed
Acetone • Rapid action
• Cheaper than ethanol
• Good for fatty tissue processing
Quickly evaporates
• Inflammable
• Prolonged use may cause shrinkage and
brittleness of tissue

11. Clearing
 Aims of clearing:
 – Removal of dehydrating agent (e.g. alcohol) to facilitate
impregnation of paraffin wax
 – To make the tissue clear and improve the microscopic
examination
 • Ideal clearing agent:
 – Low viscosity and high penetration rate
 – Low melting point
 – Miscible with both alcohol and molten wax
 – No tissue damage
 – Less toxic
 – Less inflammable
 – Cheap

12.  Selection of appropriate clearing agent: Type of tissue,
type of processor, processing condition (such as heat,
vacuum) safety factors and cost
 Volume of clearing agent: 40 times the volume of the
specimen
 • Total duration:
 – Smaller biopsy: 1 h
 – Larger tissue: Three changes in xylene or toluene
60 min each
 • End point detection: Tissue becomes transparent
 • Prolonged exposure to clearing agent: The brittle and
more friable tissue
 • Different clearing agents: Xylene, toluene, chloroform,
amyl nitrate, cedarwood oil and limonene

13. Comparison of clearing agents
Properties Xylene Toluene Chloroform Esters
Tissue
shrinkage
Yes Yes Minimum No
Tissue
hardening
Yes No No No
Inflammable Yes Yes No Yes
Harmful effect Irritant but less
harmful
Irritant Dangerous
toxic gas
liberates in
heating
Safe
Cost Cheap Cheap Very expensive High cost

14. Aims: To provide support to the tissue.
Principle: Clearing agent is removed by the process of
diffusion, and the tissue space is now infiltrated with the
embedding media.
Ideal impregnating medium:
 • Miscible with clearing agent
 • Liquid in higher temperature and solid in room
temperature
 • Homogenous and stable
 • Non-toxic and cheap
 • Transparent
 • Fit for sectioning the tissue

15. Infiltration and Embedding
 1. Size of tissue: Thicker large tissue takes more time to
impregnate with the embedding medium.
 It also contains more clearing agent to remove.
 2. Type of tissue: Hard tissue such as bone and cartilage
takes more time for embedding than soft tissue.
 3. The type of clearing agent: Certain clearing agents are
easy to remove than others. Such as xylene and toluene
are easy to remove than cedarwood oil.
 4. Type of processing: Vacuum embedding enhances
impregnation.
 Ester wax, polyester wax, resins, agar, gelatin, celloidin.

17. Equipments & Reagents
 Tissue cassettes
 Automatic Tissue Processor
 Glass jars
 Incubator
Reagents Required:-
 Ethanol
 Xylene
 Paraffin
 Isopropenol

18. Influencing Factors of Tissue Processing
Size of the tissue:
 – The smaller the size, the better the processing.
Agitation:
 – Agitation facilitates the contact of tissue with fresh
solution.
Heat:
 – Increases the better penetration of flui
Viscosity:
 – The higher the viscosity of the medium, lower the
penetration.
Negative pressure:
 – Negative pressure removes trapped air in the tissue.
 – Removal of clearing agent by increasing volatility.

19. Dehydration
 In paraffin tissue processing various dehydrating agents used
are alcohol ethanol, methanol, Isopropenol. butyal alcohol &
acetone.
 Before taking the tissue for processing it is necessary to
remove fixative from the tissue.
 After proper fixation tissue should be washed under running tap
water & then kept in 70% alcohol for minimum 30 minutes
which helps to remove fixative from the tissue.
 It is also called as “resting stage” in processing.
 If tissue will be directly introduced to high grade of alcohol
after fixation it will usually show high grade of shrinkage due to
rapid removal of water.
 After this tissue is introduced to ascending grades of alcohol &
final 2-3 changes of absolute alcohol. Between transfer of
graded alcohol tissue should be allowed to drain the previous
solution.

20. Dehydration
Procedure
1. automatic tissue
processor
a. overnight
2. Baths: water,
70,95,100,100 %
alcohol
3. Clearing agent: 2
baths of xylene

21. Clearing
 Various clearing agents used are :- chloroform, Benzene,
Xylene, Cedar wood oil.
 Clearing is done to remove the dehydrating solution & to
make the passage clear for impregnating medium to
infiltrate in the tissue.
 The clearing agent should be miscible with both the
dehydrating agent & impregnating agent.
 Many of clearing agent have a similar refractive index to
that of protein constituents making the tissue translucent .
 The end point of clearing can be noted by the transparent
appearance of the tissue after xylene treatment.

22. Impregnation with wax
 Once clearing is over the
tissues are transferred to
melted paraffin wax bath
for impregnation replacing
the clearing agent with the
embedding medium.
 We use paraffin wax of
melting point 60-62OC, so it
will remove bubbles &
replace xylene with
paraffin.
 Two baths of melted
paraffin each of 1.5 hrs & 2
hours are given.

23. Factors affecting the tissue processing:-
 Heat:- Instead of processing the tissue at RT the processing
of tissue done at 370 C will help for rapid dehydration which
in turn will help to reduce to total processing time.
 Agitation:- Continuous agitation will help for rapid
dehydration.
 Vacuum:- These will increase the speed of penetration of the
reagent which in terms will help for rapid & good quality
processing.
 Size of tissue & volume of reagent should be appropriate.
Overcrowding of tissues in the container will results in
insufficient processing.

24. Procedure for loading the tissue Processor
 All the cases from gross room are tide in cassettes, plastic
metal bought into machine room where they are kept under
running tap water for 5 minutes.
 Then these cassettes are dipped in 70% Isopropenol till the
machines are switched on.
 Then according to order of utilization of machines cases are
put into corresponding tissue baskets. These tissue baskets
are then loaded into respective machines.
 Entry of this is maintained in processing register , machines
are switched on at around ______
 The temperature of paraffin bath _____? is noted in register.
 The processed tissue is embedded in next morning.

25. Tissue Embedding

26. Introduction
 The processed & & impregnated tissues need firm
supporting embedding medium . This enables us to
have thin sections on the microtome.
 Criteria for Good Embedding medium
1. It should offers the advantages of fast embedding
2. It should give good consistency of serial sections
3. It should have wide range of section thickness
4. It should produce blocks which are durable for long
time.

27. Embedding Medium
1. Paraffin wax is a most commonly & widely
using embedding medium
2. It is a hydrocarbon produced in cracking mineral
oils , the melting point ranging between 40 to 70 0
c.
3. We use paraffin wax, of a melting point 58 to 60 o
C which is commercially added with resins.
 Praplast is highly purified paraffin wax which
produces thin sections & is mainly to cut lymph
nodes.

28. Principle
 The tissue is blocked by transferring it from
the final wax bath to a moulds filled with
molten paraffin wax & oriented on the mold in
such away that the surface to be cut rests on
the base of the mould.
 The block is then quickly cooled.

29. Materials required
1. Stainless steel metal moulds of different sizes- many small lab. Uses
“L” (Leukhartz) mould – mace up of brass
2. Plastic embedding rings.
3. Pointed forceps.
4. Lead pencils
5. Register
6. Pair of tongues
7. Gas Burner
8. Cardboard boxes
9. Small plane labels scissors.
10. Rough knives, Enamel tray
REAGENTS Required:-
 Embedding machines.
 Hot plate
 Paraffin Dispenser
 Cooling Plate

30. Procedure
1) L mould is placed on a metal sheet. Molten wax is poured with the help of
metal kettle. Individual tissue bit is placed at bottom & pressed with help
of forcep.
2) The corresponding label is attached at the side the mould. This is allowed
to cool. Moulds are taken out & the slab is cut around bits.
3) Many large laboratories having large work load are using Tissue
embedding system. This system comprise of three . i.e. wax dispenser
cold plate & heating storage area for moulds.
4) Many large laboratories use metal moulds & plastic cassettes are made in
the same plastic cassette used for processing.
5) The label with appropriate pathology number is affixed on the plastic
rings

32. Orientation of Tissues
 Skin:- must be oriented in such a way that the epidermal edge , sub
cutaneous tissue & deeper layers of all should be seen in a finished
slide. Epithelial edge should be paralleled to the cutting edge at the
upper side of the block while cutting so that cutting knife comes first
in contact with the soft part & then hard part. This gives minimum
tension on knife & we get a uniform section.
 Bone Marrow:- is placed horizontally ; parallel to the cutting edge so
that it is easy to get whole section.
 Stomach:- it should embedded in such a way that all layers i.e.
mucosa , submucosa , muscularis & serosa should seen in sectioning.
 Cervix:- the flat surface of cervix should be at bottom of the mould
so that ectocervix , endocervix & transformation zone should come
first in final slide.
 Lymphnode :- should placed at the same level, only a few mm
appear with parallel placement.
 If the mucosa is not seen microscopically the block should be melted
and tissue should be rotated through 900 & then re-embedded.

33. Points to remember
 Care is taken that the blocks are not over
cooled or else they will crack & the
sectioning becomes difficult.
 The use of over heated forceps should as
it burns the contact area of the tissue.
 Over heated paraffin also should not used.
 Do not overheat rough knife as it will burn
the label & make it unreadable.

34. Trimming & Cutting

35. Introduction
 Microtome is a mechanical device for cutting
thin uniform slices of tissues , thin enough for
examinations under a microscope.
 Prior to sectioning paraffin block should be
trimmed so that the tissue is fully exposed. The
requirement for section cutting are a well
maintained microtome sharp knife & properly
processed tissue.

36. Different types of Microtomes:-

37. Sledge Microtome
1. In this type, knife is fixed & tissue block
moves.
2. These are generally used to cut sections
of larger blocks.
3. These microtome are generally heavy.

38. The Rocking Microtome:-
 The name of this type of microtome is based
on it’s rocking action of the cross arm.
 In this type knife is fixed at one place & block
moves.
 This is generally used to cut small tissues.

39. Sliding Microtome
 This type of microtome is generally used to cut
block of celloidin material.
 In sliding microtome , knife moves horizontally
& blocks remain fixed at one place.

40. Rotary Microtome
 The name of this microtome is based on it’s rotary
action of hand wheel.
 These microtomes are heavier & stable.
 This type of microtome is used for cutting all types of
blocks. & is commonly used in histopathology
laboratories.

41.  Freezing Microtome:- This is used to cut
Frozen section specifically

42. Microtome Knife
Microtome knives are used to cut thin & serial sections of paraffin block
containing tissue Bite.
 Types of knives:-
1) Plano concave i.e profile ’A’
2) Biconcave ( Profile B)
3) Wedge (Profile C):- As
4) Tool Edge (Profile D)
 Disposable blades:- They are most popular, are of two types – low profile
& high profile.
 Advantages of disposable blades:-
1) Sharpening time & abrasive coast is saved.
2) Very thin sections are possible with this blades.

43. Knife Angles:-
For cutting thin section it is important to
know what a knife angle is !
1) Bevel Angle:- It can varied between 18
to 35 degree. It is formed by
convergence of two planes.
2) Angle of Clearance:- This angle is formed
between knife edge & block surface. It is
around 2 to 5 degrees.

44. Knife sharpening
Old method of sharpening is manual sharpening. It involves two process –
Honing & Stropping.
1) Honing :-
 It is the process in which all the nicks & irregularities in the cutting
edge of the knife are removed to make the cutting edge straight &
sharp.
 After prolonged use or after cutting very hard tissue the cutting edge
will have become so damaged that stropping will not re sharpen it ;
 small pieces of metal may have been removed which will give a
jagged edge, causing lines or tears in section cutting.
 A straight cutting edge & the correct bevel must be restored by
grinding the knife on a hone.
Types of hones are :- Belgian black vein/Belgian yellow , Arkansas ,
Carborundum
Grads of honing material :- 1. Coarse :- This type of particles are used for
quick sharpening of badly damaged knives, but we should careful as it
causes nicks in knife. 2. Fine

46. Lubricants
 They are used in sharpening knives.
 They acts as coolant & protects the temper of
extreme edge.
 They allow the flow away of fine metal
particles from the knife edge & movement of
fresh abrasive particles towards the knife edge.
 They reduces the tendency of stones pores to
become blocked with finely divided metal
particles.
 Material used are aqueous lubricants 1% liquid
soap , 50% glycerol & 10-30% soluble oil etc.

47. Precaution taken during honing
 Lubricants must use.
 Blades must kept perfectly flat while honing
because if the knife edge be raised even the
slightest , the edge will be turned.
 After honing is complete the knife should wiped
with piece of soft cloth moistened with Xylene.
 The edge may be view with low power objective
microscope to ascertain removal of nicks.

48. Stropping
•It is the process of polishing the already fairly
sharpened edge after honing
•Stropping removes the buffs formed during honing.
•The knife edge cannot be sufficiently sharp to cut
good sections directly from honing or sharpening.
•Stropping must follow sharpening. This is the final
technique for sharpening the knife before cutting
sections.

49. Types of strops:
1. Rigid / fixed – Rigid type is preferred by many
histotechnologists as it is easy to manipulate and rounding of
the knife edges is minimized.
2. Flexible / hanging
Strop is made from rump of horse and is fixed on wooden piece
with handle of the size of Length - 12 inch, Breadth - 2 inch,
Height - 2 inch.
Back of strop is made of canvass to give support to leather during
stropping.
Small quantity of vegetable oil is applied beneath the leather to
keep the strops soft

51. Section Cutting
 Clinical diagnosis need to be confirmed by laboratory investigations
for a proper patient management and therapy.
 For Histopathological diagnosis, a good thin tissue section is very
important.
 Well fixed paraffin embedded block are pre requisites for getting thin
sections. To get thin good quality tissue section for staining by histo
technologist and histopathological examination by the
histopathologist.
 Instruments required:
1. Good microtone
2. Sharp microtone knife
3. Properly processed tissue
4. Block holders
5. Water bath
6. Slide warmer
7. Forceps
8. Number 0 brush
9. Albuminized slides
10. Experience

52. Trimming of Tissue Blocks
 When everything has gone well in fixing, dehydrating,
infiltrating the specimen and embedding it, you should have
obtained a paraffin block containing the specimen surrounded
with –generally- a too generous amount of paraffin wax.
 The surplus paraffin needs to be removed and the paraffin
block trimmed in its final form prior to sectioning.
 Trimming removes excess paraffin from tissue & tissue
surface is exposed properly o get complete section of tissue.

53. PROCEDURE
1) After trimming, blocks as well as knife are
cooled on ice tray for 10-15 minutes.
2) The block is clamped firmly to the
microtome block holder.
3) Stage, blade or knife holder & block holder
clamps all should be enough tight.
4) Adjust microtome knob as per desired
microns.

54. Procedure
5) Rotate the wheel slowly with rhythmic movement till the
ribbon of sections is obtained.
6) Select section in cut from the ribbon & taken in a jar
containing water & diluted alcohol. Major folds are removed
from the section.
7) The section is now taken on albuminized slide & it is dipped
in water bath to remove microscopic folds. This is called
spreading of sections. Temperature of water bath is maintained
at 57-580.
8) The section is blotted on filter papers & pathology number is
scratched on the glass slide. These unstained slides are kept in
an oven for heating at 60 0C.

56. Points to Remember
1. Use knife guards to prevent knife injury.
2. Use Needle for Scalpel to lift ribbon from blade.
3. Knives should be handled carefully. When not in
use, it should be kept in a knife box.
4. Knife should be cleaned with xylene after every
use.
5. Do not allow the edge of knife to touch any surface.
Separate knife should be used to cut bony tissue.