2. Introduction to histopathology
Greek word Histo- tissue Pathos- disease suffering
refers to the microscopic examination of tissue in order to
study the manifestations of disease.
histopathology refers to the examination of a biopsy or
surgical specimen by a pathologist.
after the specimen has been processed and histological
sections have been placed on glass slides.
3. Collection & Transport of biopsy specimen
Surgical specimens (after removal) should be placed in
adequate quantity of fixative (10% formal saline) as soon
as possible.
For optimal fixation, a piece of tissue should be immersed
in at least 10 times of its own volume.
4. Jar or bottles with screw tops and of a suitable capacity should be
used.
Large specimens to be transported as such to the laboratory without
delay in a bucket or another suitable container.
Amputed limbs may be wrapped in a rubber sheet.
Bulky, solid specimens e.g. large tumors, spleen etc should be
bisected before placed in fixative.
Hollow viscera should be opened at both ends or cut open along
their length.
If jars are not available, the specimen should be taken fresh to the
laboratory or wrapped in moist cotton wool and be refrigerated
overnight.
5. Rapid frozen section
If a rapid frozen-section is required, the laboratory staff
and pathologist must be notified 1 hour prior to operation
but, preferably, on the preceding day.
6. Request Forms
If more than one biopsy is taken from the same patient at
the same time, a single request form is sufficient.
If two apparently unrelated pathological lesions are taken
than a separate request form should be provided with each
specimen.
Request form must contain patient’s data, lab history,
clinical data related to present illness.
In surgical cases, nature of operation and brief description
of specimens should also be provided.
7. Labelling of a specimen
Medical officer is responsible for checking the correct
labeling on the specimen.
This labeling should match with particulars present on
request forms.
8. Histotechnology
Histological technique dealing with the preparation of
tissues for microscopic examination.
A good histological technique is aimed to preserve the
microscopic anatomy of the tissues and make them hard,
so that thin sections can be made.
After staining these sections should represent the anatomy
of the tissue as closely as possible to their structure in life.
This is achieved by passing the total or selected part of the
tissue through a series of processes.
11. Introduction
As soon as cells or tissues are removed from the body they
begin to die and undergo post- mortem changes.
These changes may be autolytic or putrefactive.
Autolysis : Is a self destructive process due to the release
of autolytic enzymes from the dead cells .
Putrefaction : Occurs due to the action of bacteria that
invade the tissue.
12. Definition of Fixative
A fixative may be defined as a substance which prevent
post mortem changes and preserves the morphological and
chemical characteristics of cells and tissues.
13. Aims of Fixation
1.It should prevent autolysis & putrefaction of the cell.
2.It should penetrate evenly and rapidly.
3.It should harden the tissues
4. Increase the optical differentiation of cells & tissues
5.Should not cause shrinkage or swelling of the cells
6.Must not react with the receptor sites & thus must not
interfere with the staining procedure.
7.It must be cheap and easily available.
14. Good fixation is most important factors in the production of
satisfactory results in histopathology.
Following factors are important:
• Fresh tissue
• Proper penetration of tissue by fixatives
• Correct choice of fixatives
No fixative will penetrate a piece of tissue thicker than 1 cm.
For dealing with specimen thicker than this, following methods are
recommended:
1. Solid organ: Cut into slices but not thicker than 5 mm.
2. Hollow organ: Either open or fill with fixative or pack lightly with
wool soaked in fixative.
3. Large specimen: It requires dissection, Inject fixative along the
vessels or bronchi as in case of lung so that it reaches all parts of the
organs.
15. Properties of an ideal fixative
Prevents autolysis and bacterial decomposition.
Preserves tissue in their natural state and fix all
components.
Make the cellular components insoluble to reagent used in
tissue processing.
Preserves tissue volume.
Avoid excessive hardness of tissue.
Allows enhanced staining of tissue.
Should be non-toxic and non-allergic for user.
Should not be very expensive.
16. Mechanism/Action of fixatives
At the molecular level, fixative have the property of
coagulating proteins in the tissue, through the formation of
crosslink's between protein molecules thereby keeping
their relation to each other.
20. Physical methods of fixation
Heat fixation : The simplest form of fixation is heat.
Microwave Fixation: Microwave heating speeds fixation and can
reduce times for fixation of some gross specimens and histological
sections from more than 12 hrs. to less than 20 min.
Freeze-Drying and freeze substitution : Freeze-Drying is a useful
technique for studying soluble materials and small molecules;
tissues are cut into thin sections, immersed in liquid nitrogen, and
the water is removed in a vaccum chamber at -40oc .
• The tissue can be post-fixed with formaldehyde.
21.
22. Chemical Fixation
Chemical fixation utilizes organic or non- organic
solutions to maintain adequate morphological
preservation.
Chemical fixatives can be considered as members of three
major categories.
1.coagulant
2.Cross-linking
3.Compound fixatives
23. Simple Fixatives
Formalin
• The most commonly used fixative is Formalin
• It is prepared by mixing 40 % Formaldehyde in 100 w/v of
distilled water.
• The resultant mixture is 100 % Formalin.
• Routinely, 10 % formalin is used which is prepared by
mixing 10 ml of 100 % formalin in 90 ml of distilled water.
24. Mechanism of action
It forms cross links between amino acids of proteins
thereby making them insoluble.
It fixes 4 mm thick tissue in 8 hours.
25. Advantages
1. Rapid penetration
2. Easy availability & Relatively cheap(Low cost)
3. Does not over harden the tissue
4. Fixes lipids for frozen sections
5. It is relatively easy to prepare
6. It allows subsequent use of most staining procedures.
7. Frozen sections can be made with formalin fixed tissue.
26. Disadvantages
1. Irritant to the nose, the eyes and mucous membranes
2. Formation of precipitate of paraformaldehyde which can
be prevented by adding 11- 16 % methanol.
3. Formation of black formalin pigment , Acid
formaldehyde hematin.
4. It causes shrinkage of collagen.
5. it suspected to contain cancer producing agents
28. 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.
29. Microanatomical Fixatives
10 % Formal saline :
It is a microanatomical fixative.
Ideal for fixation of brain.
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.
30. Fixatives used for Electron Microscopy
Glutaraldehyde
Osmium tetroxide
Formaldehyde & Glutaraldehyde mixture
Acrolein
31. Factors affecting fixation
1. Temperature
2. Size of the specimen
3. Volume ratio
4. Duration of Fixation
5. Choice of fixatives
6. Penetration
7. Tissue Storage
8. Buffer & pH
9. Osmolality
Editor's Notes
As biopsy- A small piece of lesions or tumor which in sent for diagnosis before final removal of the lesion or the tumor (Incisional biopsy).
If the whole of the tumor or lesion is sent for examination and diagnosis by the pathologist, it is called excisional biopsy.
The frozen section is the rapid tissue section by cooling the tissue with the help of cryostat to provide immediate report of the tissue sample. The cryostat is the instrument to freeze the tissue and also to cut the frozen tissue for microscopic section.
