Examination of Fresh Tissue and Fixation Flashcards
The microscopic study of the normal tissues of the body
Histology
The microscopic study of tissues affected by disease
Histopathology
The simplest, 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
Removes not only cells, but also a small portion of the surrounding tissue. This provides 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 noy all. The doctor will slice into the lesion and remove only a portion of it. If the lesion is found to be cancerous, further surgery may be needed to remove the lesion.
Incisional biopsy
Generally removes the entire area in question
Excisional biopsy
Considered to be the primary technique for obtaining diagnostic full-thickness skin specimens
Punch biopsy
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 or spooned to remove tissue or growth from body cavity such as endometrium or cervical canal
Curettings
specimens that are usually examined when there is an immediate need for evaluation
Fresh tissues
Is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as normal saline solution or roger’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 or dissociation
In teasing or dissociation, the specimen is either stained with _____ or examined unstained by _______
supravital
phase-contrast microscopy
Is a process whereby small pieces of tissue (not more than one mm in diameter) are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass.
Squash preparation (Crushing)
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
the method of preparing the smears differs depending on the nature of the material to be examined.
Smear preparation
These may be examined as either as fresh prep similar to that described for teased preparations, or by using a supravital technique.
Smear
This is useful for preparing smears of thick secretions like mucous fluids, serous fluids, sputum, enzymatic lavage samples from GI tract, and blood smears
Smear preparation
This is useful for preparing smears of thick secretions like mucous fluids, serous fluids, sputum, enzymatic lavage samples from GI tract, and blood smears
Smear preparation
With an applicator stick or an 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.
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
It is little more tedious than streaking, but has many advantage of maintaining cellular interrelationships of the material to be examined
Spreading
It is specially recommended for smear preparation of fresh sputum and bronchial aspirates, and also for thick mucoid secretions
Spreading
This is done by placing a drop of secretion or sediment upon one slide and facing it unto another clean side.
Pull-apart
This is a special method of smear preparation whereby the surface of a freshly cut pieces 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)
A fresh tissue is frozen on a ______ with ____
microtome with CO2
A cold chamber kept at an atmospheric temperature of ______.
Cryostat
-10C to -20C
Commonly used methods of freezing include:
- Liquid nitrogen
- Isopentane cooled by liquid nitrogen
- Carbon dioxide gas
- Aerosol sprays
Freezing agent generally used in histochemistry and during intra-operative procedures.
Liquid nitrogen
The most rapid of the commonly available freezing agents.
Liquid nitrogen
The main disadvantage of a liquid nitrogen is that soft tissues are liable to crack due to rapid expansion of ice within the tissue, producing ______
ice crystals or freeze artifacts
_______ is liquid at room temperature
Isopentane
Tissue blocks can also be frozen by adapting a conventional freezing microtome gas supply of _______ from a ______
Carbon dioxide gas
CO2 cylinder
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)
Is a refrigerated apparatus used for fresh tissue microtomy
Cryostat or Cold Microtome
Cryostat consists of microtome (rotary microtome) kept inside a cold chamber w/c maintains the temperature between _______ by an adjustable thermostat
-10 to 1-30C (ave. -20C)
Majority of the sections in cryostat can be cut in _____, where the temperature for sectioning can be accurately established and controlled.
Isothermic situations
Capable of freezing fresh tissues within 2-3 minutes
Thermostat
Cutting sections of Crytostat
4 micra
Provides sections for fresh tissue examination esp. Fluorescent Antibody Staining techniques or Histochemical enzyme studies
Cryostat or Cold Microtome
It is the first and most critical step in the histopathology
Fixation
Primary aim of fixation
Preserve the morphology and chemical integrity of the cell
fixation must preserve:
shape
structure
intercellular relationship
chemical constituents
fixation must prevent:
degeneration
decomposition
putrefaction
distortion of tissues
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
2 mechanisms in fixation:
- Additive fixation
- Non-additive fixation
Mechanism of fixation where chemical constituents are taken in and become part of the tissue. It form cross-links and stabilize protein
Additive fixation
Mechanism of fixation where the fixing agent is not incorporated into the tissue. Removing of water to form new cross-links
Non-additive fixation
Main factors involved in fixation:
- Hydrogen-Ion Concentration
- Temperature
- Thickness of section
- Osmolality
- Concentration
- Duration of fixation
Fixation is best carried at pH ____
pH 6-8
Surgical specimens must be at _______
room temperature (20-25C)
Tissue processors must be maintained at ____
40C
Electron microscopy must be at ______
0-4C
Formalin at 60C is used for _____
urgent biopsy specimens
Formalin at 100C is used to ____
fix tissues with tuberculosis
High temperature –> ____ Distortion
Higher
Thickness of section for electron microscopy
1-2 mm2
Thickness of section of light microscopy
2 cm2
Thickness of section for light microscopy (thin sections)
0.4 cm
Brain tissue is fixed using _____ for 2-3 weeks for easier cutting of sections
10% Formalin
Hypertonic solution may lead to _____
shrinkage
_______ may lead to swelling and subsequent poor fixation
Hypotonic and Isotonic solution
Osmolality normal val:
400-450 mOsm
Osmolality of Isotonic solutions
340 mOsm
Recommended/Best Osmolality
Slightly Hypertonic solutions
Most common fixative:
10% neutral buffer formalin
Fixing agent used in electron microscopy
3% Glutaraldehyde
Fixing agent used in Immunoelectron microscopy
0.25% Glutaraldehyde
Presence of this causes polymerization of the aldehyde –> decreased effectiveness
BUFFER
Recommended time for primary fixation in buffered formalin from the time the specimen is obtained
2-6 hours
Recommended time in electron microscopy for duration of fixation
3 hours
Average penetration:
1 mm/hour
