SAS 3: Examination of Fresh tissue and Fixation Flashcards
It is the microscopic study of the normal tissue of the body
Histology
It is the microscopic study of tissue affected by disease
Histopathology
The simples, least invasive test and uses the smallest needle to simply remove cells from the area of abnormality
Fine needle aspiration
This is not always adequate to obtain a diagnosis, depending on the area being biopsied
Fine needle aspiration
remove not only cells, but also a small portion of the surrounding tissue
Core needle biopsy
This provide additional information to assist in the examination of the lesion
Core needle biopsy
takes out even more surrounding tissues. it takes some out of the abnormality, but not all
Incisional biopsy
The doctor will slice into the lesion and remove only a portion of it. if the lesion is found cancerous, further surgery may be needed to remove the lesion
Incisional biopsy
generally removes the entire area in question
Excisional biopsy
Considered the primary technique for obtaining diagnostic full-thickness skin specimen
Punch biopsy
it requires basic general surgical and suture tying skills and is easy to learn
punch biospy
the technique involves the use of a circular blade that is roated down through the epidermis and dermis, and into the subcutaneous fat, yielding a 3-4 mm cylindrical core of the tissue sample
Punch biopsy
where small fragments of tissue are shaved from a surface (usually a skin)
Shave biopsy
where tissue is scooped and spooned to remove tissue or growth from body cavity such as endometrium or cervical canal
Curettings
Fresh tissues have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as
Motion
Mitosis
Phagocytosis
is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as normal saline solution or Ringer’s solution in a petri dish or watch glass, carefully dissected with a needle and separated by direct or zigzag spread using an applicator stick
Teasing or dissociation
is a process whereby small pieces if tissue (not more than 1 mm in diameter) are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass
Squash preparation (crushing)
this method differs depending on the nature of the material to be examined
Smear preparation
may be examined as either as fresh prep similar to that described for teased preparation, or by using a supravital staining technique
Smear
with an applicator stick or platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion.
Streaking
A selected portion of the material is transferred into a clean slide and gently spread into moderately thick film by teasing the mucous strands with applicator stick
Spreading
This is done by placing a drop of secretion or sediment upon one slide and facing it unto another clean slide.
Pull-apart
this is a special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination phase-contrast microscopy or staining for light microscopic study
Touch preparation (Impression smear)
Immediate diagnosis is accomplished
Frozen section
it is necessary to get a rapid diagnosis of pathologic process
Frozen section
A fresh tissue is frozen on a microtome with CO2, or on a cryostat, The cold chamber is kept at an atmospheric temperature of
-10C to -20C
frozen section, both fixed and unfixed, have many applications in histotechnology, and are commly used for:
- undehydrated tissues for rapid diagnosis
- histological demonstration of fats
- For certain neurological structures
- for sensitive tissue constituent damage by heat
- diagnostic and research enzyme histochemistry
- immunohistochemical and immunofluorescent staining
commonly used method of freezing include:
- liquid nitrogen
- Isopentane cooled by liquid nitrogen
- Carbon dioxide gas
- Aerosol sprays
generally used in histochemistry and during intra-operative procedures, and is the most rapid of the commonly available freezing agents
Liquid nitrogen
its main disadvantage is that soft tissue is liable to crack due to rapid expansion of ice within the tissue, producing ice crystals or freeze artifacts
Liquid nitrogen
Is a refrigerated apparatus used for fresh tissue microtomy
Cryostat or Cold microtome
It has a rotary microtome kept inside a cold chamber
Cryostat or Cold microtome
the cold chamber of Cold microtome must maintain the temperature of
between -10 to -30 (average -20C)
Also used for intraoperative diagnosis
Cryostat or Cold microtome
It is the first and most critical step in the histopathology
Fixation
What is the primary aim of Fixation
To preserve the morphology and chemical integrity of the cell
Fixation must preserve:
- shape
- structure
- Intercellular relationship
- Chemical constituent
Fixation must prevent:
- Degeneration
- Decomposition
- Putrefaction
- Distortion of tissues
What are the Objectives of fixation
- To preserve the tissue
- To prevent breakdown of cellular elements
- To coagulate or precipitate protoplasmic substances
two mechanisms in fixation:
- Additive fixation
- Non-additive fixation
Chemical constituent are taken in and becomes part of the tissue
Additive fixation
Fixing agent is not incorporated into the tissue
Non-Additive fixation
The Main factors involved in fixation
- Hydrogen ion concentration
- Temperature
- Thickness of section
- Osmolality
- Concentration
- Duration of fixation
Fixation is best carried at what pH
pH: 6-8
Surgical specimens must be at what temperature
Room temperature (20-25C)
Tissue processor must be maintain at what temperature
40C
Electron microscopy is at what temperature
0-4C
Formalin at 60C is used for _____
urgent biopsy specimens
Formalin at 100C is used to ____
fix tissues with tuberculosis
Thickness of section in Electron microscopy
1-2mm^2
Thickness of section in light microscopy
2cm^2
Thickness of section in light microscopy (thin section)
0.4 cm
brain tissue is fixed using ____ for ____ weeks for easier cutting of section
- 10% formalin
- 2-3 weeks
Most common fixing agent used
10% formaldehyde
fixing agent Used in electron microscopy
3% glutaraldehyde
fixing agent used in immunielectron microscopy
0.26% glutaraldehyde
recommended time for primary fixation in buffered formalin from the time the specimen is obtained
2-6 hours
Recommended time of Electron microscopy for duration of fixation
3 hours
Traditional volume of the tissue
10-25x the volume of the tissue
The volume of maximum effectiveness
20x the tissue volume
Fixation can be improved by the ff:
- Heat
- Vacuum
- Agitation
- Microwave
Characteristics of a good fixative
- must be cheap
- must be stable
- must be safe to handle
- must kill the cell quickly
- must inhibit bacterial decomposition and autolysis
- must produce minimum shrinkage of tissue
- must permit rapid and even penetration of tissue
- must harden tissues
- must isotonic
- must make cellular components insoluble to hypotonic solutions
- permit application of many staining procedure
In teasing or dissociation, the specimen is either stained with _____ or examined unstained by _______
supravital
phase-contrast microscopy
In squash preparation (crushing), a supravital stain if necessary, may be placed at the junction of the slide and the cover glass, and allowed to be absorbed by the tissue through _____
capillary attraction
A fresh tissue is frozen on a ______ with ____
microtome with CO2
_______ is liquid at room temperature
Isopentane
The use of this has become increasingly popular in recent years, and is adequate for freezing small pieces of tissue except muscle.
Aerosol sprays
Quick-freezing spray cans of _____ have a distinct advantage of rapidly freezing blocks of any type of tissue
Fluorinated hydrocarbons (Cryokwik)
Majority of the sections in cryostat can be cut in _____, where the temperature for sectioning can be accurately established and controlled.
Isothermic situations
Leaving the tissue specimen in ____ will cause to dry out and result to distortion
Air
Leaving the tissue in water (hypotonic solution) will cause the cell to _____
swell
Strong salt (_____) will cause the cell to ____.
Hypertonic solution
shrink
High temperature –> _____ distortion
Higher
Hypertonic solution may lead to _____
shrinkage
_______ may lead to swelling and subsequent poor fixation
Hypotonic and Isotonic solution
Osmolality normal value:
400-450 mOsm
Osmolality of Isotonic solutions
340 mOsm
Recommended/Best Osmolality of the Specimens
Slightly Hypertonic solutions
Most common fixative:
10% neutral buffer formalin
Average penetration:
1 mm/hour
