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Histopathology
- Examination of Fresh Tissue
- Tissue Processing
- Fixation
- Fixatives

Types of smear preparation




Examination of Fresh Tissue
Surgical Procedures:






Fine needle aspiration
- least invasive test, simplest
- remove cells from the area of abnormality
Core Needle biopsy
- Removes not only cells, but a small amount of
the surrounding tissue
...









Excisional Biopsy
- Removes the entire area in question
...

Shave biopsy
- Small fragments of tissue are "shaved" from a
surface, usually skin
...


Methods of Fresh Tissue Examination
1
...

> View cells using phase contrast/ Brightfield
Microscope
2
...

> A vital stain is applied (selectively stains living cells)
3
...




Streaking
> Using an applicator stick or a platinum
loop, the material is rapidly and gently
applied in a direct or zigzag line
...
Material disperses evenly
over the surface of the two slides
...

> Used for thick secretions such as serous
fluids, concentrated sputum, enzymatic
lavage (GI tract) and blood smears
...
Touch Preparation (Impression smear)
> Special method of smear preparation whereby the
surface of a freshly cut piece of tissue is brought into
contact and pressed on to the surface of a clean glass
slide, allowing cells to be transferred directly to the
slide for examination by Phase Contrast Microscopy
...
Frozen Sections
> Most ideal and preferred method
> Rapid diagnosis of a pathologic tissue during
surgery
...

> Cryostat: Optimum working temperature -18-20 C
Methods of Freezing
- Liquid Nitrogen (Most rapid freezing agent)
- Isopentane cooled by liquid nitrogen
- CO2 gas
- Aerosol sprays



Tissue Processing
“F DCIETS SML”
1
...

(Decalcification) – Optional
 Removal of calcium
 Specimens for: bones, teeth, etc
...

2
...
Clearing/ Dealcoholization
 Process of replacing the dehydrating fluid
with a fluid that is miscible with BOTH the
dehydrating fluid and the
impregnating/embedding medium
4
...

5
...
Trimming
 Process of removing excess wax after
embedding
 Ideal: Four-sided prism/ Truncated pyramid
7
...
5 um
8
...

 Different kinds of stain:
> Hematoxylin and Eosin (Histopathology)
> Gram’s stain and Ziehl-Neelson
(Microbiology)
> Romanowsky (Hematology)
> Papanicoloau (Cytolygy)
 Natural Dyes
> Hematoxylin
> Cochineal dyes
> Orcein
> Saffron
 Synthetic Dyes
> AKA “Coal tar dyes”
>Derived from benzene and collectively
known as Aniline dye
9
...
518)
- Eukitt
- XAM (1
...
532)
- Canada Balsam (1
...
544)
> Aqueous Media (usually for lipids because
resinous media contain xylene which may
dissolve in fats)

- Glycerin (1
...
43)
- Karo corn syrup
- Apathy’s med
...
52)
- Brun’s fluid: recommended for mounting
frozen sections from water
...
Labeling
 Label for the department, unique number,
and year
 Example: SP – 001 – 22

FIxatives



Classification of Fixatives
 Physical fixative
 Heat
 Freezing
 Chemical Fixatives
Simple fixatives
 Aldehydes
□ Formaldehyde
□ Glutaraldehyde
 Metallic fixatives
□ Mercuric chloride
□ Chromate fixatives
 Picric acid
 Acetone
 Acetic acid
 Alcohol
 Osmium tetraoxide
 Osmic acid
 Compound fixatives
 Microanatomical fixatives
 10% formal saline

 10% neutral buffered formalin
 Heidenhain's Susa
 Formal sublimate (formal
corrosive)
 Zenker's solution
 Zenker-formal (Kelly's solution)
 Bouin's solution
 Brasil's solution
 Cytologic Fixatives
 Nuclear
□ Flemming's fluid
□ Carnoy's fluid
□ Bouin's fluid
□ Newcomer's fluid
□ Heidenhain's Susa
 Cytoplasmic
□ Flemming's fluid without
acetic acid
□ Kelly's fluid
□ Formalin w/o post-chroming
□ Orth's fluid
□ Regaud's fluid (Muller's fluid)
 Histochemical
□ 10% formal saline
□ Absolute ethyl alcohol
□ Acetone
□ Newcomer's fluid

Aldehyde Fixative
 Formaldehyde
 37-40% stock solution
 Liquid form
 Distilled water as diluent
 10% working solution
 Advantage: cheap, easy to prepare
and readilyavailable
 Disadvantage: May produce
considerableshrinkage of
tissues
 May cause brittle tissues
 10% Formol Saline
 For CNS tissues
 Advantage: Preserves enzymes
and nucleoproteins, demonstrates
fats and mucin
 Disadvantage: slow fixative
 10% buffered Neutral Formalin
 For post-mortem and research
specimen

 Advantage: Best fixative for tissue
containingiron pigments
 Disadvantage: It is longer to prepare
...

 Heidenhain's Susa
 For tumor biopsies of the skin
 Adv: penetrates and fixes
tissues rapidlyand evenly
 Disadv: RBC preservation is poor
 B5 Fixative (B5BMB)
 For bone marrow biopsies
 Adv: rapid fixation can be
achieved in 1 1/2
- 2 hrs
 Disadv: Over fixation hardens
tissue

 Picrates are formed upon protein;
precipitates aresoluble in water;
 Hence tissues must be first rendered
insoluble bydirect immersion in 70%
ETOH
 Picrates fixative must never be
washed in waterbefore dehydration
 70% ethyl alcohol - 5% sodium thiosulfate
- runningwater
 Bouin's solution
 For embryos and pituitary biopsies
 Adv: excellent for preserving soft
and delicatetissues
 Disadv: penetrates larges tissues poorly
 Brasil's solution (BRAniGLYCO)
 For excellent fixative for glycogen
 Adv: less messy

Glacial Acetic Acid (GAA-Nucleo)
 Adv: Fixes and precipitates nucleoproteins
 Disadv: destroys mitochondria and golgi
apparatus

Alcoholic
Fixative
 Methyl alcohol
 For dry and wet smears
 Isopropyl alcohol
 For fixing touch preparations
 Ethyl alcohol
o 70-100%
 Carnoy's (fastest Car)
 most rapid fixative, fixation time 1-3hrs
 Newcomer's fluid

 Fixes mucopolysaccharide

Osmium Tetraoxide
 Should be kept in a dark-colored
chemically cleanbottle to prevent
evaporation and reduction by sunlight
or organic matter
 Inhibits hematoxylin and makes
counterstainingdifficult
 Produces black precipitate (osmic oxide)
 Prevention: add saturated aqueous HgCl2
 Remedy: dissolve in cold water
 Flemming's
 For nuclear structure
 Flemming's with acetic acid
 For cytoplasmic structures

Trichloroacetic acid
 Both a fixative and decalcifying agent

Acetone (Asotone)
 Fixes brain tissues for the diagnosis of rabies

Heat Fixation
 Direct flaming
 For bacterial smear
 Microwave fixation
 Optimum temperature: 45-55C



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