2. CONTENT
Introduction
Steps In Histopathology
1.Preparation of sample 2.Processing of sample
A. Sample collection A. Dehydration
B. Fixation B. Clearing
C. Infilteration
D. Embedding
E. Sectioning
D. Staining
Storage of paraffin blocks
3. INTRODUCTION
• Histo pathology is an Greek word.
• Histo – Tissue pathology – disease suffering.
• Histopathology is the Department of Clinical Laboratory which deals with the
study of different types of Tissues.
• Examination of biopsy or surgical speciamen by a pathologist.
4. STEPS IN HISTOPATHOLOGY
1. PREPARATION OF SAMPLE
A. Sample Collection
• Tissues (with patient history)
• Bones (with X-ray)
• Autopsy (consent form)
• Body fluids
• CSF (Cerebrospinal fluid )
• Skin tissues
• Etc……
B. Fixation
• A Chemical process by which biological tissue are preserved from decay either
through autolysis or putrefaction.
5. • Samples were collected and processed by ‘’FORMALIN’’. Formalin is commonly
used fixative in all laboratories.
Commonly used fixatives in laboratory
1. 10 % Formalin
2. Formal Saline
3. 10 % Buffered Formalin
4. Bouins Solution
5. Alcoholic Formaldehyde
6. Zenkers Ffluid
6. 2.PROCESSING OF SAMPLE
A. Dehydration
• Removal of fixative and water from tissue.
• In most instances, starts by placing in 70% ethanol progressing
through 95% to 100% ethanol.
• For delicate tissues (sembryonic tissues), start with &
80% ethanol
• Dehydrating agent should not be less than 10 times the volume of
the tissue
Commonly used dehydrating agents
1. Alcohol
2. Acetone
3. Diethylene dioxide
7. B. Clearing
• Clearing is required to remove alcohol from tissues and is replaced by fluid which is miscible with wax with
which tissue must be impregnated
• Clearing agent is miscible with both dehydrating agent as well as paraffin wax
Commonly Used Clearing Agents In Laboratory
• Benzene
• Xylene
• Toluene
• Choloroform
• Petroleum ether
• Oil of wintergreen (Methyl salicylate)
• Cedar wood oil
• Carbon tetrachloride
• Dioxane
• Aniline oil
8. C. Infilteration /Impregnation
• Infiltration/impregnation. The role of the infiltration agent is to remove the
clearing agent from the tissue and to completely permeate the tissue with
paraffin wax.
• This will allow the tissue to harden and produce a wax block from which thin
histological sections can be cut.
Commonly used infilteration medium
Paraffin is the most commonly used medium for infiltration, embedding and
formation of tissue blocks.
9. D. Pre embedding / Embedding
• Once tissue samples are infiltrated by paraffin they are removed from the
casettes and carefully positioned inside a metal base mold.
• The mold is filled with melted paraffin and then immediately placed on a
cooling surface.
• In this manner the cassette will be used as a base of the paraffin block of
microtome sectioning.
E. Sectioning
• The paraffin block is mounted on the microtome holder.
• Sections are cut as a ribbon and floated on hot water bath maintained at 45*C
to stretch the paraffin section.
• Tissue section are then allowed to dry at room temperature or 37*c.
10. • At the end of the processing the sample is inserted into molds and covered with liquid paraffin.
• Then, the cassette with the identification number of the patient and additional paraffin is placed
on the mold.
• The mold is then laid on a cold surface in order to favor its solidification and subsequent
creation of the block.
Types Of Mold
1.Paper mold
2.Metal mold
3.Dimmock mold
4.Disposable mold (plastic)
5.Metal or plastic mold used for ring cassettes (F) or standard cassettes (G)
11.
12. F. Staining Methods
1. PAP staining
• This method is used to differentiate cells in the smear preparation of various gynecological specimens
(pap smears), materials containing exfoliative cells and material from fine needle aspiration.
• Papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the basic components of the
cell and basic dye stain the acidic components of the cell.
• The polychromatic PAP stain involves five dyes in three solutions.
1. Hematoxylin
2. Orange Green 6
3. Eosin Azure
4. Light green SF
5. Bismarck brown Y
• Nuclei : Blue
• Acidophilic cells : Red
• Basophilic cells : Blue Green
• Erythrocytes : Orange-red
13. 2. Hematoxylin and Eosin (H&E) staining:
• This is a classic standard tissue section staining method widely used for the
inspection of tissue components for pathological analysis that’s applicable in
all organs and disease models.
• based on binding of nucleic acid and other acidic components of the tissue to
the basic hematoxylin stain.
The acidic counter stain, eosin, binds to basic components in the tissue, such as
cytoplasmic proteins.
3. TBS (Toluidine blue staining):
• This method is used for staining of mast cells that are found in the connective
tissue and their cytoplasm contains granules composed of heparin and
histamine.
• Following Toluidine staining, mast cells are stained red-purple and the
background is stained blue
14. STORAGE OF TISSUE SLIDES OR PARAFFIN BLOCKS
• The tissue slides or paraffin blocks are store with sufficient space between them in a dry
and reasonably air tight boxes in a fridge (4*C) for a month.
