This document discusses tissue processing and fixation. It begins by introducing tissue fixation and its objectives, such as preventing degradation and maintaining morphology. It then describes various fixation methods and factors that affect fixation quality. Common fixatives are discussed, including formaldehyde, glutaraldehyde, Zenker's solution and Bouin's solution. Fixation protocols for specific tissues like brain, breast, lung and kidney are also reviewed. The document emphasizes the importance of proper fixation for histological examination.

1. TISSUE PROCESSING:
TISSUE FIXATION
AND
GROSSING
Presented By:
Dr. Akash

2. CONTENTS
• Introduction
• Tissue process in pathology
• Objective of fixation
• Properties of fixative
• Mechanism of fixation
• Fixation artifacts
• Types of fixation: physical and chemical
• Factors affecting fixation
• Fixation for electron microscopy
• Fixative for individual tissues
• Disposal of tissues and fixatives
• Grossing /surgical cut-up

3. INTRODUCTION
• Fixative may be defined as a substance which prevent
post mortem changes and preserves the morphological
and chemical properties of cells and tissues.
• Appropriate fixation of tissues for histological
examination is central to all histological studies.
• Without this all tissues will degrade and analysis will
be useless.
• The mechanism and principals by which specific
fixative acts fall into several broad groups: covalent
addition of reactive groups and cross links,
dehydration, affects of acids, salt formation and heat.
• Several of these mechanism may work together.

4. Type of material obtained in laboratory:
• The human tissue comes from the surgery (Biopsy)
and/or from the dissection room (Autopsy).
• From surgery two types of biopsy could be obtained:
• Incisional Biopsy: A small piece of lesions or tumor
is removed and sent for diagnosis before final removal of
the lesion or tumor .
• Excisional Biopsy: whole tumor or lesion is removed
for examination.

5. Tissue processes in pathology

6. OBJECTIVES OF FIXATION (why fixation
is done)
• Stops degradation of cellular components by enzymes.
• Prevent autolysis and putrefaction.
• Maintain clear and consistent morphological features
and original microscopic relationships between cells and
its components.
• Local composition of the tissues should also be
maintained.
• Water soluble component of the tissues should not be
lost during fixation.
• HARDENING AND SOLIDIFICATION: converts the
semifluid consistency of cells into an irreversible
semisolid consistency, hardening allows easy
manipulation of soft tissues.

7. • Optical differentiation: Fixatives alter to varying degrees
the refractive indices of the various components of cells
and tissues and thus increase its optical differentiation.
• Effects of staining: Certain fixatives like formaldehyde
intensifies the staining character of tissue especially with
haematoxylin.

8. Properties of an Ideal Fixative:
1. Prevents autolysis and bacterial decomposition.
2. Preserves tissue in their natural state and fix all
components.
3. Make the cellular components insoluble to
reagent used in tissue processing.
4. Preserves tissue volume.
5. Avoid excessive hardness of tissue.
6. Allows enhanced staining of tissue.
7. Should be non-toxic and non-allergic and non
corrosive for user.
8. Should not be very expensive.
9. It must penetrate the tissue rapidly and evenly
10. It must be simple to prepare and economical to
use

9. MECHANISM OF FIXATIVES
PROTEINS:
• Most fixative act by denaturing
or precipitating proteins.
• These proteins then form a
sponge or meshwork to holds
the other constituents.
• At the molecular level fixative
have the property of
coagulating proteins in the
tissue through formation of
cross links.
• Formaldehyde -reversible.
• Glutaraldehyde -rapid &
irreversible.

10. Nucleic acid:
• Fixation brings a change in the physical & chemical state
of RNA & DNA.
• Uncoiling of DNA & RNA occurs with formalin when
heated to 45˚C & 65˚C respectively.
Lipids:
• Phospholipids are fixed by aldehydes.
• Formaldehyde reacts with unsaturated fatty acids hence
less lipid can be demonstrated in tissue stored in it for a
long time.
• Mercuric chloride reacts with lipids to form complexes.
• Ultrastructural demonstration of lipids – post fixing in
imidazole-osmium tetroxide.

11. Carbohydrates:
• Carbohydrates are more water soluble- difficulty in total
preservation
• They bind with fixed protein
• So the fixatives which are used for proteins, can be used
for carbohydrate preservation.
• Fixed protein traps carbohydrates.
• Glycogen not bound to protein- fixed protein form lattice
around glycogen to preserve it
• Glycogen are more demonstrable in liver cells

12. Fixation artifacts
Fixation is associated with some artifacts:
• Formaldehyde fixatives give brown pigmentation
to tissues.
• Mercuric chloride fixatives leave a black
precipitate in tissues.
• Some fixatives produce shrinkage in tissues while
some others cause swelling.
• Due to poor penetration of fixatives
macromolecules like glycogen diffuse from
unfixed parts giving false localization or loss. This
is termed streaming artifact and mostly seen in
case of glycogen.

13. TYPES OF FIXATION
• There are two broad categories of fixation.
1. Physical method of fixation
2. Chemical method of fixation
Physical methods are independent processes and are not used
commonly in the routine practice of medical histology.
Most methods of fixation used in the processing of tissue for
histopathological diagnosis rely on chemical fixation.

14. 1. PHYSICAL METHOD OF FIXATION
• Following methods are included:
1. Heat fixation
2. Microwave fixation
3. Freeze-drying and freeze substitution

15. 1.HEAT FIXATION:
• This is the simplest form of fixation.
• Boiling of the egg is the perfect examples of the
process.
2.MICROWAVE FIXATION:
• Microwave fixation speeds up fixation and can reduce
the time for fixation of some gross specimens from
more than 12 hrs to less than 20 mins.
• Microwaving the tissue in formalin can produce
potentially explosive vapours which may cause safety
problems.

16. 3.FREEZE-DRYING AND FREEZE SUBSTITUTION:
• Freeze drying is a useful technique for studying
soluble materials and small molecules.
• Tissues are cut into sections, immersed in liquid
nitrogen, and the water is removed in a vacuum
chamber at -40oC.
• Tissues can be post fixed with formaldehyde
vapours.
• Bring the temperature gradually upto 4oC to
complete the fixation process.

17. 2. CHEMICAL FIXATION
• This utilizes organic or non-organic solutions to
maintain adequate morphological preservation.

18. • Chemical fixatives can be considered as members of
three major categories..
i. Coagulant
ii. Cross linking
iii. Compound fixative

19. SIMPLE FIXATIVES
• FORMALINE:
• It is most commonly used fixative
• Prepared by mixing 40% formaldehyde gas in 100 w/v of
distilled water.
• This will produce 100% formaline.
• Routinely 10% formaline is used which is prepared by
mixing 1oml of 100% formaline in 90ml of DW.

20. • MECHANISM OF ACTION:
• Forms cross links between amino acids of proteins
thereby making them insoluble.
• It fixes 4mm thick tissue in 8hrs.
• ADVANTAGES:
• Rapid penetration
• Easy availability and low cost
• Doesn’t over hardens the tissue
• Relatively easy to prepare
• It allows subsequent use of most staining procedures.

21. • DISADVANTAGES:
• Irritant to eyes and respiratory
epithelium
• Formaline dermatitis
• Cause shrinkage of collagen
• Formation of precipitate of
paraformaldehyde
• Formation of black formalin
pigment, acid formaldehyde
hematin. (avoided by using buffered
formaline)
• Brown, granular material,
extracellular, birefringent

22. Other Simple fixatives:
• Glutaraldehyde
• Osmium tetraoxide: demonstrates myelin
• Pottasium dichromate: fix cytoplasm and mitochondria
• Mercuric dichromate
• Mercuric chloride
• Picric acid:enhance cytoplasmic staining
• Zenker’s fluid
• Bouin’s fluid

23. COMPOUND FIXATIVES
Microanatomical fixatives:
• These are used to preserve the anatomy of
• the tissue.
Cytological fixatives:
• These are used to fix intracellular structures.
Histochemical fixatives :
• These are used to demonstrate the chemical constituents
of the cell

24. • Microanatomical Fixatives:
1. 10 % Formal saline :
• It is a microanatomical fixative.
• Ideal for fixation of brain.
2. Buffered formalin:
• Due to the presence of buffer, the pH of the solution
remains at neutral or near neutral.
• As a result, Formalin pigment formation doesn’t take
place

25. Cytological Fixatives:
Nuclear fixatives :
• Carnoy’s Fluid
• Clarke’s Fluid
• Newcomer’s Fluid
• Flemming’s Fluid
Cytoplasmic Fixatives :
• Champy’s Fluid
• Regaud’s Fluid
Special Fixatives:
• Dichromate and chromic acid fixatives

26. Histochemical Fixatives:
• Formal saline
• Cold acetone
• Absolute alcohol

27. Composition & Preparation of
Fixatives:-
1- 10%Formalin Solution :
• It is recommended for fixation of general surgical
• biopsy specimen and tissues from CNS.
• It causes even fixation and very little shrinkage because
of its isotonicity.
• Composition & Preparation:
• Formaldehyde (37-40%) - 10 ml
• Distilled water - 90 ml
• Mix well.

28. 10% Neutral Buffered Formalin Solution:
• It is recommended for research specimens but may also
be used for surgical and post mortem specimens.
• The special advantage of NBF is that it prevent the
formation of troublesome acid formalin pigment .
Disadvantage :
• Laborious and time consuming
Composition & Preparation:
• Formaldehyde (37-40%) - 100 ml
• Distilled water - 900 ml
• NaH2PO4 - 4.0 g
• Na2HPO4 (anhydrous) - 6.5 g
• Mix to dissolve.

29. Zenker's Solution:
• M.C. Fixatives rapidly penetrates tissues and permit
excellent staining of nuclei and connective tissues.
• fixation time 4-24 hours.
• Disadvantage : causes hardening of tissues
• Composition & Preparation :
• Distilled water - 950ml
• Potassium dichromate - 25g
• Mercuric chloride - 50g
• Glacial acetic acid - 50g
Fixed tissue should be washed overnight in running tap
water before processing.

30. Bouin's fluid:
• Fixation time 6 hours.
Composition & preparation:
• Saturated aqueous solution of picric acid - 75ml
• Formalin (~ 40% aqueous solution of formaldehyde) - 25ml
• Glacial acetic acid - 5ml
Fixed tissue should be transferred to 70% alcohol.
Carnoy's fluid:
• Fixation time 1-3 hours.
Composition & Preparation:
• Ethanol - 60ml
• Chloroform - 30ml
• Glacial acetic acid - 10ml
Fixed tissue should be processed immediately or transferred to
80% alcohol.

31. FACTORS AFFECTING FIXATION
• Temperature: most tissues fixed at room temperature, for
electron microscopy and histochemistry 0 to 4oC is used.
• Size of the specimen: bulky specimens take more time to fix
• Volume ratio
• Duration of Fixation: formaline takes 4 to 6 hrs, for electrone
microsopy tissue should be fixed for 3hrs.
• Choice of fixatives: Adding substances to fulfill certain functions.
Denaturing effects, some stabilize proteins. Eg. Sodium chloride &
sodium sulphate used with mercuric chloride. Tannic acid
enhances fixation of lipids & proteins in EM
• Penetration
• Tissue Storage
• Buffer & pH
• Osmolality: best: slightly hypertonic solution

32. Fixatives used for Electron
Microscopy:
• Glutaraldehyde: most efficient cross linking agent
for collagen, more rapid fixation than formalin
But gives false positivity with PAS
• Osmium tetroxide
• Formaldehyde & Glutaraldehyde mixture
• Acrolein

33. Fixatives for DNA, RNA, and protein analysis:
• The compounds included the commercially
available HOPE (Hepesglutamic acid buffer
mediated Organic solvent Protection Effect) for the
detection of nucleic acids, in addition to zinc-based
fixatives.
• In contrast to other fixation methods HOPE does
not completely denature or cross-link structural
proteins, enzymes, and nucleic acids.
• They remain in an almost native state. This means
that HOPE- fixed tissue can also include active
viruses, prions, microorganisms etc.

34. Fixation for selected individual tissues:
Eyes:
• The globe must be firmly fixed in order to cut good sections
for embedding.
• Eyes may be fixed in NBF usually for approximately 48 hours.
Brain:
• The problem of fixing a whole brain is to make it firm enough
to investigate the neuroanatomy and to be able to produce
sections for histopathology and possible immunochemistry.
• Conventionally this fixation takes 2-6 weeks.
• To speed up the process perfusion of the brain via the middle
cerebral arteries done, bypassing the slow rate of fixative
penetration from the outside.
• Fixation may also be enhanced by the use of microwave
technology.

35. Breast:
• Clinical samples should be fixed in 10% NBF for a
minimum of 6–8 hours, to a maximum of 72 hours.
• The time from the tissue acquisition to fixation should be
as short as possible in order to prevent lysis of clinically
important biomarkers such as estrogen receptors,
progesterone receptors and the human epidermal growth
factor receptor-2 (HER2)
• If the tumor specimen has come from a different
geographical location to the laboratory, it should be
bisected through the tumor on removal from the patient
and sent to the laboratory immersed in a sufficient
volume of NBF.

36. Lungs:
• Lung biopsies are typically fixed in NBF. The lungs from
lobectomy, pneumonectomy and autopsies may be inflated by,
and fixed in NBF instilled under gentle pressure via the
trachea or major bronchi.
• Such fixed lungs can be cut within 2-6 hours, and gross
sections are fixed overnight, allowing sections to be processed
and cut the next day.
Lymphoid tissue:
• Special care should be taken with all lymphoid tissue as many
organisms, e.g. Mycobacterium tuberculosis and viruses may
be present in the lymphoreticular system. There is always a
possible infection risk with such cases.
• The lymphoid tissue is usually sliced and a representative
sample of fresh tissue taken for special studies, e.g.
microbiology, flow cytometry or molecular analysis. The rest
of the lymph node is fixed in NBF.

37. Muscle biopsies:
• The tissue for routine histological assessment is fixed in NBF
and embedded so the fibers of the specimen are viewed in
crosssection and longitudinally.
• After processing this is stained with H&E, a trichrome stain
and Congo red if amyloid is suspected.
Renal biopsies:
• Renal core biopsies should be subdivided into three and each
piece should contain an adequate number of glomeruli, e.g. 6-
10 for light microscopy, 1-2 for electron microscopy and 3-6
for immunofluorescence.
• Each portion is then preserved depending upon the method to
be used for subsequent analysis:
•i NBF for routine histology.
•ii Buffered glutaraldehyde at pH 7.3 for ultrastructural
analysis.
•iii Snap frozen in isopentane and liquid nitrogen for
immunofluorescence examination.

38. Disposal of fixatives and tissues
• Tissues are potentially infectious and care must be taken in
handling and disposing for the safety of self and staff.
• Fixative and chemicals can not be disposed into the general
waste system(i.e, down sink drains)
• All fixatives must be placed into special designated containers
for disposal.
• Mercury containing fixatives must be disposed of according to
institutional and legal standards.
• Xylene and methanol must be disposed into special waste
containers. Xylene is a neurotoxin and short term exposure can
cause headaches, dizziness, lack of coordination, confusion and
fatigue.
• Clean ethanol can be disposed into sink drains.
• Tissues and explanted synthetic materials are discarded into
biohazard bags that are incinerated.

39. GROSSING/SURGICAL CUT-UP
Specimen reception:
• A separate room is required for specimen reception must be
equipped with appropriate easily cleaned benching, adequate
lighting, good ventilation, safety equipment, disinfectants,
absorption granules and protective clothing.
• The key point of this room is to receive samples safely and
securely.
• Any new specimen should have its identity confirmed and
assigned a unique laboratory specimen identifier, usually a
complex number.
• Mapping of the specimen identifier against the clinical
request form is mandatory, along with checking of
appropriate clinical details mentioned against the specimen.

40. • Corroborative data, in the form of the hospital
number/registration index, unique patient identifier
number, the full name, date of birth and address are
valid ways of verifying the identity of any specimen.
• The usual numerical method of specimen identification
is simply the year, expressed in two digits, with a
sequential numbering system starting with one (1) and
proceeding up to the final specimen of each year.

41. Surgical cut-up/specimen dissection/Grossing:
• The dissection area must have good electrical or
natural lighting, good ventilation and non-absorbent
wipe-clean surfaces.
• There must be facilities available that the
pathologist can do the dissection either by standing
or sitting way.
• Various types of cutting instruments should be
available so that the pathologist can choose the
instrument according to the tissue size and texture
and other requirements.

44. Thinking before dissection:
• The way the tissue sample taken depends upon the
type of tissue and the lesion presenting with it, eg:if
any growth appears in any specimens of GB then it
mandates to take the tissue samples from that area.
• This dissection/blocking/grossing/cut-up facility
must have an appropriate storage area immediately
to hand allowing clearance of already-examined
samples promptly, preventing the dissecting area
becoming cluttered.
• The blade must be sharp if one is going to
confidently sample the tissue appropriately to
produce blocks of the correct thickness and shape.

46. Photography:
• Photographing the macroscopic specimen, whole or
during the dissection, is particularly important in cases
of complex surgical excision, e.g. Wertheim’s
hysterectomy, pneumonectomy and localization
samples.

47. • It may also be of use in later analysis/ case
discussion
• Helps in retrospective case reviews.
• professional photography may still be required
however for cases which may be used in visual
teaching presentation, journal/book publication or
in a medico-legal situation.

48. Specimen dissection plans:
• Small specimens need not to undergo any dissection and
can be processed as they present ie whole ambedded.
Core biopsies:
• Smaller biopsies are prosessed as such without any
dissection.
• Larger cores with diameter of 4-5mm or greater may be
divided into 2 halves along the long axis.
Bowel specimens:
• Bowel samples are generally medium and large sized
tissue resections.
• They are best sampled by multiple blocks of any lesion in
relation to adjacent mucosa, wall and serosal aspect of
the tissues.

49. Lung tissue:
• The lung samples commonly received for histology
are localized, wedge biopsies or lobectomy and
pneumonectomy specimens.
• The background pleura and lung must be evaluated
along with any lesions
• In general terms, multiple blocks for any tumor (4
or more) along with sampling of the
pleural/mediastinal/bronchial margins are needed.
• Careful dissection of the hilar tissues should allow
further node harvest from these tissues

50. Gynecological samples:
• Samples include fragments of endometrium removed by
curette or equivalent and small punch biopsies. These are
generally embedded whole and processed in one cassette.
• More complex samples, e.g. cone biopsies from the cervix,
need appropriate inking of margins and orientation, often in a
serial block fashion across the specimen with photography.
This allows the three-dimensional assessment of dysplasia or
invasive neoplasia in relation to the various surgical margins.
• Uterine samples are usually sampled in terms of the cervix if
included, endometrium and myometrial tissues together with
some representative sampling of common benign lesions, e.g.
fibroids (leiomyomas).
• Dysplastic and malignant lesions often require multiple blocks
including resection margins and careful examination of related
lymph nodes
• Specific tissues such as tubes and ovaries should follow similar
standard guidelines in terms of the sampling pattern, number
of blocks and related tissue samples.

51. Breast resections:
• Multiple blocks of the tumor are usually required. The
background tissues should also be assessed at multiple
points and the lymph nodes, if present, are often examined
in a tiered or grouped fashion.
• This allows the tumor spread to be assessed by identifying
the size of the nodes involved and the furthest node from the
primary which has been affected.
Soft tissue resections:
• Careful slicing and examination of the specimens
macroscopically will allow sampling and consideration of all
the peripheral boundaries.
• Furthermore, given the pervasive nature of soft tissue
tumors this widespread sampling is usually required.
• Tumor sampling before fixation for molecular and/or
genetic analysis may be required.