This document discusses the process of histopathology specimen processing. There are several key steps: 1) Tissue specimens are fixed, typically in formalin, to preserve their structure. 2) The tissues then undergo dehydration using increasing concentrations of alcohol, followed by clearing with xylene to remove the alcohol. 3) The tissues are then infiltrated with paraffin wax through impregnation to harden them. 4) The tissues are embedded in paraffin blocks. 5) Sections are cut from the paraffin blocks using a microtome and placed on slides. The slides are then stained to visualize the tissue structures under a microscope.

1. 1 1
Histopathology specimen processing
‘’Tissue processing’’
By
William Edema
Arua Institute of Health Science

2. Contents
• Introduction
• Specimen Accessioning
• Gross Examination
• Tissue Processing steps:
• The paraffin Technique and its alternatives
• The freezing Technique
• Problems in tissue processing

3. Introduction
There are 3 main techniques which are used in
preparing microscopical sections from tissues:
• The paraffin technique (the most common method)
• The celloidin technique (the most perfect method)
(read about this one!!)
• The freezing technique (the most rapid method)

4. …Introduction
• Tissues from the body taken for diagnosis of disease
processes must be processed in the histology laboratory
to produce microscopic slides that are viewed under the
microscope by pathologists.
• The techniques for processing the tissues, whether:
• Biopsies,
• Larger specimens removed at surgery, Or
• Tissues from autopsy, are described below.
• The persons who process the tissues and make the glass
microscopic slides are HISTOTECHNOLOGISTS.

5. Biopsies:
• Incisional biopsy: performed prior to major surgical
procedure when removal of entire lesion is impossible
• Purpose: strictly a diagnostic nature.
• Excisional Biopsy: In this technique, the entire lesion is
removed, usually with a rim of normal tissue.
* It is performed when the lesion is smaller in size.
* The procedure serves diagnostic and
therapeutic function.
• Core Needle Biopsy: done with special type of wide
bore biopsy needle.
* permits a percutaneous approach to internal
structures

6. Specimen Accessioning
• Tissue specimens received in the
surgical pathology laboratory have
a request form that lists the
patient information and history
along with a description of the
site of origin.
• The specimens are accessioned by
giving them a number that will
identify each specimen for each
patient.

7. Information which must be provided
 Patient Identification
 Identification of individual requesting examination
 Procedure date
 Adequate clinical history and physical examination
 Organs resected or biopsied
 Orientation if required.
 Intraoperative findings
 Prior surgery or pathologic diagnosis
 Prior treatment
 Mention if rapid diagnosis is required.

8. Gross examination
• On arrival to the lab, the tissues are macroscopically
examined by a pathologist or pathology assistant, a
processing referred to as ‘grossing’.
• Gross examination consists of describing the
specimen and cutting and placing all or parts of it
into a small plastic cassette which holds the tissue
while it is being processed to a paraffin block.
• Initially, the cassettes are placed into a fixative.

9. Grossing: Tissue must arrive
to lab in a fixative
Grossing is an art:
A knowledge of what needs be
taken for microscopic study is crucial
for final diagnosis.

11. …Gross examination: Inking
• When a malignancy is
suspected, then the specimen is
often covered with ink in order
to mark the margins of the
specimen.
• Different colored inks can be
used to identify different areas
if needed.
• When sections are made and
processed, the ink will mark
the actual margin on the slide.
Why ink:
• Resection margins
• Embedding instructions
• Orientation
• Distinguish between
samples
• Identify the cut surface

12. General principles of grossing
• Specimen identification
• Identify all the anatomical structures present
• Orientation markers should be identified
• Measurements: Length, breadth, height
• Weight
• Examine the external surface.
• Cut all the organs at intervals of 1cm thickness
• Describe cut surface, identify pathologic process
• If suspected lesion is present, measure, describe with
colour & consistency.
• Surgical margins

13.  Biological tissues must undergo a series of treatments
to be observed with light and electron microscopes.
 The process begins by stabilization of the tissue with
chemical fixatives.
 Next, the tissue is made rigid to allow sectioning.
 Finally, it is stained to provide contrast for
visualization in the microscope.

14. Tissue Processing steps
• Biological tissues are generally rather soft, making
it quite difficult to cut acceptably thin sections
directly from the fresh or fixed tissues.
• Methods must be used to hold the tissues firm, which
facilitates cutting thin sections with a sharp knife.
• Firmness can be achieved either by embedding the
tissues in a suitable embedment or by freezing the
tissue.
• Once the tissue has been fixed, it must be processed
into a form in which it can be made into thin
microscopic sections.

15. …Tissue Processing steps
• The usual way this is done is with paraffin.
• Tissues embedded in paraffin—which is similar
in density to tissue, can be sectioned at anywhere
from 3 to 10 microns, usually 5-8 routinely.
• The technique of getting fixed tissue into paraffin
is called tissue processing.
• The main steps in this process are dehydration
and clearing.

16. • Paraffin-embedded tissue processing therefore describes
the steps required to take tissue from fixation to the state
where it is completely infiltrated with a suitable
histological wax and can be embedded ready for
sectioning
• Designed to remove all extractable water from tissue,
replacing it with support medium that provide sufficient
rigidity to enable sectioning of the tissue without
damage or distortion
TISSUE PROCESSING
1. The paraffin Technique and its alternatives

18. Decalcification
• Decalcification (if required) is done between
fixation and dehydration. It is the process of
removal of the calcium salts from the specimen.
• The various agents used for decalcifying are:
• Nitric acid
• Hydrochloric acid
• Formic acid
• Picric acid
• Acetic acid
• Citric acid

19. Tissue processing

20. Tissue processing….
• Can be performed manually: Not convenient and
efficient for multiple specimens:
–use an automated “tissue processor”.
• There are two main types of processors:
– tissue-transfer (or “dip and dunk”) type
– fluid-transfer (or “enclosed”) type
• Most modern fluid-transfer processors employ raised
temperatures, effective fluid circulation and
incorporate vacuum/pressure cycles to enhance
processing and reduce processing times.

21. Tissue Processing Machine
• Factors affecting
rate of processing:
• Agitation
• Heat
• Viscosity
• Pressure

22. Overnight processing schedule
Station Reagent Time Temp
1 10% formalin 1 hour 38 degree C
2 10% formalin 1 hour 38 degree C
3 50% alcohol 1 hour 38 degree C
4 70% alcohol 1 hour 38 degree C
5 90% alcohol 1 hour 38 degree C
6 90% alcohol 45 min 38 degree C
7 100 % alcohol 1 hour 38 degree C
8 100 % alcohol 45 min 38 degree C
9 Xylene 1 hour 60 degree C
10 Xylene 30 min 60 degree C
11 Paraffin 1 hour 60 degree C
12 Paraffin 1 hour 60 degree C

23. 1. Tissue fixation
• Crucial step in tissue preparation
• Purpose: prevents tissue autolysis
and putrefaction.
• Therefore, transfer samples
into fixative immediately after
collection.
• The optimum formalin-to-
specimen volume ratio should be
at least 15-20:1
• This will allow most tissues to
become adequately fixed within
24-48 hours.

24. 1. Fixation…
• Most commonly used reagent for the fixation is 10% neutral
buffered formalin
• Formalin containers should be capped and leak-proof, and labelled
correctly.
• The tissue bits should be of the size ~2x2cm & 2-3
micrometer in thickness.
• These bits are then placed in metal cassettes or capsules which
are then placed in the fixative.
• Tiny biopsies or small specimen can be wrapped in a filter paper
and then put in a cassette & fixed.
• To help in visualization of small fragment of tissue during
embedding, a few drops of 1% eosin is added

25. Types of fixatives
• Physical methods:
– Heating, Microwaving, Freeze drying
Chemical methods
• Coagulatant fixatives :(Precipitate and coagulate protein)
– Alcohols and acetone
– Picric acid and trichloro acetic acid
• Cross linking fixatives:
– Formaldehyde and glutaraldehyde
– Other aldehydes (chloral hydrate, glyoxal)
– Metal salts (Mercuric and zinc chloride, osmium
tetroxide)
• Compount fixatives

26. Physical fixation
• Microwave irradiation fixes the tissue more
effectively
• Large specimens which are soft to cut, can be
irradiated for a brief period & then the tissue
blocks are cut.
• The procedures of dehydration & impregnation is
accelerated.
• Tissue antigens are well preserved following
microwave irradiation.
• Microwave processing can be applied in
electron microscopy

27. Other Fixatives
• Glutaraldehyde and Osmium Tetraoxide
• B5 fixative:
– stock solution: Mercuric chloride, sodium
acetate & distilled water
– Add 2ml of formaldehyde to 20ml of stock
solution
• Zenker's fluid
• Bouin’s Fluid

28. Specimen Transfer to Cassettes
• After fixation, specimens are trimmed using a
scalpel to enable them to fit into an appropriately
labelled tissue cassette.
• Specimens should not be so big that they fill the
cassette
– they are trimmed so as not to touch the edges.
• Additionally, they must not be too thick, otherwise,
they risk being “waffled” when the cassette lid is
closed.
• The filled tissue cassettes are then stored in formalin
until processing begins.

29. The paraffin Technique
Washing
Following fixation, the tissues should be washed
for 15 to 30 minutes. The fixed tissues are washed
in running tap water to remove the fixative from
them.

30. 2. Tissue Processing
Processing tissues into thin microscopic sections is usually
done using a paraffin block, as follows:
• Dehydration: involves immersing specimens in increasing
concentrations of alcohol to remove the water and formalin
from the tissue.
• Clearing: organic solvent e.g. xylene is used to remove the
alcohol and allow infiltration with paraffin wax.
• Embedding: specimens are infiltrated with the embedding
agent – usually paraffin wax.
• The tissue becomes surrounded by a large block of molten
paraffin wax, creating what is now referred to as the
“block” providing a support matrix

31. • Wet fixed tissues (in aqueous solutions) cannot be directly
infiltrated with paraffin.
• First, the water from the tissues must be removed by
dehydration.
• Dehydration must be done gradually e.g. with a series of
alcohols; say 70% to 95% to 100% to prevent turbulence
at the interface between water and pure ethanol. Turbulence
cause damage or distortion to cellular components.
• The number of steps or the gradient differences should be
determined by
• The degree of fixation
• The delicacy of the tissue
• The degree of cellular detail to be preserved
• Sometimes the first step is a mixture of formalin and alcohol.
2 (a) Dehydration

32. 2 (a) Dehydration
• Dehydrating agents are strong hydrophilic, possessing
strong polor groups that interact with water by hydrogen
binding.
• Most common dehydrant used is alcohol
• Other dehydrants can be used, but have major disadvantages.
• Acetone is very fast, but a fire hazard, so is safe only for small,
hand-processed sets of tissues.
• Dioxane can be used without clearing, but has toxic fumes.
• Ethano acetone
• Isopropyl alcohol
• Glycol

33. •The next step is called "clearing" and consists of removal of the
dehydrant with a substance that will be miscible with the embedding
medium (paraffin).
•Clearing agents therefore act as intermediary infiltration solution
between dehydration & embedding medium
• They provide translucent appearance to the tissue
• Choice of a clearing agent depends upon the following:
• Rapid penetration of tissue
• Speedy removal of dehydrating agent
• Ease of removal by melted paraffin wax
• Minimal tissue damage
• Safety factors (low fammability, low toxicity)
• Cost and convenience
2 (b) Clearing

34. 2 (b) Clearing
• The commonest clearing agent is xylene.
• Other Clearing agents include: toluene, chloroform, methyl
salicylate
• Toluene works well, and is more tolerant of small amounts
of water left in the tissues, but is 3 times more expensive
than xylene.
• Chloroform used to be used, but is a health hazard, and is
slow.
•
• Methyl salicylate is rarely used because it is expensive, but it
smells nice (it is oil of wintergreen).
• Excessive exposure to clearing reagents may cause excessive
hardness or shrinkage.

35. • Impregnation is the process of infiltrating or allowing
the paraffin penetrate in-between the cells of the tissues.
• This process of paraffin infiltration is a necessary step
to harden the tissues before their embedding.
• Here, the tissues are put for 6 – 24 hours in hot soft
paraffin at 50°C, then in hot hard paraffin at 55 °C in
the oven.
• Infiltration must be carried out at only a few degrees
above the melting point of paraffin
2 (c) Impregnation in paraffin

36. Paraffin wax
• Most popular infiltration and embedding medium
• It is a mixture of long chained hydrocarbons produced by mineral
oil
• It permeates the tissue in liquid form and solidify rapidly when
cooled
• The paraffin wax increases the optical differentiation, hardens
the tissue & helps in easy sectioning of the tissue.
• It is compatible to most routine, special stains as well
as immunohistochemistry protocols
• Alternative embedding media: Resin, gelatin and celloidin
2 (c) Impregnation in paraffin

37. • Finally, the processed tissue is to be correctly oriented
and enclosed in a embedding or support medium that
provide external support during microtomy.
• The embedding agent is almost always paraffin.
• Paraffin is a derivative of crude petroleum. It is a
group of variable length, long-chain hydrocarbons of
the methane series
• Most paraffins suitable as embedding media melt
between 52° and 58°C.
• Since most paraffin have a melting point between 52-
58°C, it must infiltrate the cells while it is hot.
3. Embedding/blocking

38. 3. Embedding/Blocking:
• Impregnated tissues are placed in a mould with their
labels and then fresh melted wax is poured in it and
allowed to settle and solidify.
• Once the block has cooled sufficiently to form a surface
skin it should be immersed in cold water to cool it
rapidly.
• After the block has completely cooled it is cut into
individual blocks and each is trimmed.
• Labels are made to adhere on the surface of the block.

39. Embedding Centre

40. Embedding process

41. 4. Microtomy/Sectioning /Section Cutting
• Microtomy is the means by which tissue is sectioned and
attached the surface for microscopic examination
• The instrument by which this is done is called as a
Microtome.
• TYPES OF MICROTOMES:
• Sliding
• Rotary
• Rocking
• Freezing
• Base sledge
• Ultramicrotome
• Microtome knives:
• Disposable blades
• Glass and diamond knives

42. Paraffin section cutting
Equipment required
• Flotation water bath
• Slide drying oven or hot plate
• Forceps, brush and teasing needles
• Clean slide
• Slide rack
• Diamond pencil
Section adhesives
• Albumin, gelatin, starch
• Poly-L-lysine
• 3-aminopropyltriethoxysilane (APES)
• Charged or plus slides (permanent)

43. 4. Sectioning
• Specimens are cut into sections that can be placed on a slide.
• Trimming: Wax is removed from the surface of the block to expose the
tissue.
• A microtome is used to slice extremely thin tissue sections off the block in
the form of a ribbon; most tissues are cut at around 5 µm.
• Floating: Tissue ribbons are carefully transferred to a warm water bath.
Here the tissues are allowed to float on the surface, and can then be
scooped up onto a slide placed under the water level.
• Charged slides work best for this process
• Allow slides to dry upright at 37oC for a few hours to gently melt the
excess paraffin wax, leaving the tissue section intact.

44. This is done by a machine called microtome.
The blocks are sliced in the range of 5 to 15 µm (1 µm =
1/1000mm)
The slices then
are fixed on the
slides.
µ
µ
4. Sectioning

45. The mold used for this specimen was
too small. The specimen is in contact
with the edges of the block and may
therefore be difficult to section.
Proper tissue size and positioning

47. 5. Staining
• Definition
• The process to stain or give color to the tissue
sections with the dyes
• The stains can be routine stain or special
Routine staining
• Hematoxylin Eosin
Specific/special staining
• Histochemistry e.g. PAS
• Immunocytochemistry

48. 5. Staining
• Most cells are transparent and appear almost
colorless when unstained.
• Stains are therefore used to provide contrast
to tissue sections, making tissue structures
more visible and easier to evaluate.
• Following staining, a coverslip is mounted
over the tissue specimen on the slide, using
optical grade glue, to help protect the
specimen.

49. Autostainer
Staining (H & E)

51. Staining
Routine staining
• The most commonly used staining in histology
• Cheapest staining
• Easy to be done
• The microstructure is stained well
• Can be studied under light microscopy
• Hematoxylin-eosin staining
• The nuclei of cells are stained blue (basophilic) in color
• The cytoplasms of cells including the organelles and
other molecule are stained pink (Acidophilic) in color

52. Specific/special staining
Histochemistry staining
The staining is used to localize specific chemical
constituents of tissues and cells
• Based on
 enzyme activity
• Example: Nitric Oxide Synthase (NOS)
 Chemical reactivity
• Example: Periodic acid Schiff (PAS) to
localized the glycogen and carbohydrate rich
molecules

53. Histochemistry staining
• Reactions of interest
 monitored by formation of an insoluble
precipitate that take a certain color
• Staining can be applied to the whole mount
or histological slide
Specific/special staining

54. Immunocyto/histochemical staining
• Definition
• A technique to identify cellular or tissue
constituents (antigens) by means of antigen-
antibody interactions.
• The site of antibody binding
• Is identified by direct labeling of antibody or by
used a secondary labeling method
• The terms
• If tissue Immunohistochemistry
• If cell Immunocytochemistry
Specific/special staining

55. Immunocyto/histochemical staining
• Antigens: A protein or carbohydrate or lipid
molecule
• Has one or more antibody binding sites
 A highly specific topographical
regions composed of a small number
of amino acids or monosaccharide unit
known as antigenic determinant groups
or epitopes
Specific/special staining

56. Immunocyto/histochemical staining
• Antibody: A molecule that can recognize and bind to the antigen
• 2 type of antibody
 Polyclonal antibody
• An antibody that can bind to several similar epitopes
• May cross react with other molecule
• Low sensitivity
 Monoclonal antibody
• An antibody which is sensitive only to one epitope
• Not cross react with other molecule
• High sensitivity
Specific/special staining

57. Staining (H & E)
Procedure
1) Deparaffinize sections (2 changes of xylene, 10min each)
2) Re-hydration (100%, 70%, 50% & 30%- 5min each)
3) Wash briefly in distilled water.
4) Stain in hematoxylin solution for 10 min.
5) Wash in running tap water
6) Differentiate in 1% acid alcohol for 30sec
7) Wash running tap water for 1min.
8) Bluing in 0.2% ammonia water for 30sec
9) Counter-stain in eosin solution for 2 minute.
10) Graded Dehydration (30%, 50%, 70% &100%- 5min each)
11) Clearing with xylene (2 times- 5 min each)
12) Mount with DPX

60. Mountants
 DPX ( Distrene Dibutyl phthalate Xylene ).
 Canada Balsam
 Colophonium resin
 Terpene resin

62. H & E stain

63. Other stains

64. Other stains

65. Other stains

66. Alternatives to paraffin embedding
• Alternatives to paraffin embedding include various
plastics that allow thinner sections. Such plastics
include:
• Methyl Methacrylate: very hard and therefore good
for embedding undecalcified bone.
• Glycol Methacrylate (GMA): has the most widespread
use since it is the easiest to work with.
• Araldite: same as methacrylate, but requires a more
complex embedding process.
• Epon: is routinely used for electron microscopy
where very thin sections are required

67. Note:
• Plastics require special reagents for dehydration
and clearing that are expensive.
• For this reason, and because few tissues are plastic
embedded, the processing is usually done by hand.
• A special microtome is required for sectioning
these blocks.
• Small blocks must be made, so the technique is
used for small biopsies, such as bone marrow or
liver.

68. The freezing Technique
• In this method, the fresh or fixed tissues are freeze hardened
and cut with a freezing microtome in the cryostat apparatus
within few minutes
• It is a quick and simple method which is commonly used
during operations for rapid diagnosis of tumors e.g. carcinoma
• The chemistry of tissues is preserved because we use no heat
and no chemical solvents
• Can be used in Histochemistry to demonstrate enzymes and
chemical components of tissues
• Disadvantage: It gives not-serial thick sections which may
fragment into small pieces, so they are very difficult to be
stained and to be stored.

69. Problems in Tissue Processing
"Floaters" are small pieces of tissue that appear on
a slide that do not belong there--they have
floated in during processing.
Floaters may arise from sloppy procedure on the
cutting bench-- dirty towels,
instruments, or gloves can have tissue that
is carried over to the next case.
Therefore, it is essential that you do only one
specimen at a time and clean thoroughly before
opening the container of the next case.