HISTOPATH GREGORIOS 40-61 Flashcards
Immediate diagnosis is accomplished through the use of a
frozen section
It is especially recommended when lipids and nervous tissue elements are to be demonstrated
Frozen section
Frozen section is especially recommended when __________ tissue elements are to be demonstrated
lipids and nervous
Frozen sections are usually done on __________ as well as on __________
muscle and nerve biopsies
surgically removed tumors
A fresh tissue is frozen on a:
microtome with co2 / cryostat (-10 to -20 deg)
What fixative is used sa frozen section
liquid fixative
The morphological detail and resolution of frozen sections are [mas maganda/mas panget] compared to that embedded in paraffin
MAS PANGET
The advantage of the frozen section method
Rapid processing time
Less equipment
Less need for ventilation
Applications of frozen sections:
Rapid diagnosis during sx
Enzyme histochemistry
Demonstration of soluble substances such as lipids and carbohydrates
Immunofluorescent and immunohistochemical staining
Silver stains, particularly in neuropathology
[REDIS]
Slow freezing can cause distortion of tissue due to
ice crystal artifacts / freeze artifacts
The more commonly used methods of freezing include:
Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays
[LICA]
The most rapid of the commonly available freezing agents
Liquid nitrogen
Liquid nitrogen is generally used in ______ and during ______ procedures
histochemistry
intraoperative
Main disadvantage of liquid nitrogen
liable to crack due to the rapid expansion of the ice within the tissue
overcools urgent biopsy blocks causing damage to both block and blade if sectioning is done at -70°C or below
The tissue snap-frozen in
liquid nitrogen must therefore be _____ before sectioning is attempted
allowed to equilibrate to cryostat chamber temperature
The majority of non-fatty unfixed
tissues are sectioned well at temperatures between:
-10°C and -25°C
One problem with the use of liquid nitrogen is that it causes a _____
vapor phase to form around the tissue
Effect of vapor phase
formation around the tissue [liquid nitrogen]
acting as an insulator that causes uneven cooling of tissue [muscle biopsy]
Ano gagawin pag may vapor phase
formation around the tissue due to liquid nitrogen?
freeze the tissue in:
Isopentane,
OCT, or
Freon 2.2
[bec of their high thermal conductivity]
Isopentane is [solid/liquid] at room temperature
LIQUID
A Pyrex glass beaker containing
isopentane is usually suspended in a flask of liquid nitrogen until _____
half-liquid and half-solid stage is reached
_____ is usually suspended in a flask of liquid nitrogen until half-liquid
and half-solid stage is reached
A Pyrex glass beaker containing
isopentane
The Pyrex beaker is removed from the liquid nitrogen when _____
small crystals start forming on the side of the beaker (approximately -170°C)
The beaker is removed from the liquid nitrogen when small crystals start forming on the side of the beaker (approximately -170°C) and the tissue to be frozen is _____
dropped into the cooled liquid isopentane
Tissue to be frozen in ispentane is affixed on a:
cork disc,
aluminum foil, or
cryostat chuck
is adequate for freezing small pieces of tissue except muscle
aerosol sprays
_____ have a distinct advantage of rapidly freezing blocks of any type of tissue
Quickfreezing spray cans of fluorinated hydrocarbons (e.g., Cryokwik)
Fresh, completely unfixed tissues, or tissues that have been briefly treated
with formalin may not require embedding anymore; instead they may be _____
frozen and cut in a freezing microtome or cryostat.
Two methods of preparing frozen
sections may be resorted to:
1. _____
2. _____
- Cold Knife procedure
- Cryostat procedure (Cold Microtome)
[COLD KNIFE PROCEDURE]
A piece of filter paper soaked in __[1]__ is placed on the microtome
stage, and short bursts of __[2]__ are applied, freezing the filter paper to the stage
- gum syrup
- co2
Thickness of tissue section sa COLD KNIFE procedure
3-5mm
Gano katagal yung short bursts ng CO2 sa cold knife procedure
1-2 seconds duration
4 second interval
the point at which sections may then be cut at 10 µm thickness
DewLine
[COLD KNIFE PROCEDURE]
The tissue is lifted up to the knife manually and trimmed until _____
the surface is flat
[COLD KNIFE PROCEDURE]
After frozen tissue surface becomes flat, it is warmed with the finger until ______
the hard frozen tissue starts to thaw and becomes visible to the naked eye
Cold knife microtomy sections do not form ribbons but rather ______
stick to the knife blade
Cold knife sections that are stuck to the knife blade must be removed with _____
a camel hair brush or finger moistened with water
Cold knife sections are then transferred to a dish of ______
distilled water to separate
[COLD KNIFE MICROTOME]
The water dish is usually placed on a
______ background
dark or black
[COLD KNIFE]
The surface of the block may then be softened by warming slightly
with the ______
ball of the finger or thumb
[COLD KNIFE]
Tissues that have not been sufficiently
frozen will cut __[1]__, and the block may __[2]__
- thick and crumble
- come away from the stage
Optimum temperature used for sectioning in COLD KNIFE microtomy
Knife = -40° to - 60°C
Tissue = -5° to - 10°C
Environment = 0° to - 10°C
[COLD KNIFE]
Sections thinner than _____ generally cannot be obtained even from tissues that section well, and with ideal conditions for sectioning
6 µ
The success of this procedure [COLD KNIFE MICROTOMY] depends upon _____
ambient temperature and
humidity
Microtome, knife, specimen and atmosphere are kept at the same
temperature
Cryostat
Microtome, knife, specimen and atmosphere are not kept at the same
temperature
Cold knife / Cold microtome
The cryostat consists of an insulated microtome housed in an ___[1]___ and maintained at temperatures near ___[2]___
- electrically driven refrigerated chamber
- -20°C
The optimum working temperature of cryostat
-18 to -20°C
Majority of the sections can be cut in ___[1]___ conditions, where the
temperature for sectioning can be ___[2]___
- isothermic
- accurately established and controlled
Fresh frozen tissue requires that the tissue be maintained in the _____ during cutting of section
frozen solid state
[CRYOSTAT]
The tissue must be sufficiently cold to prevent ______of cell and tissue structures as the knife passes thru it.
compression and displacement
[cryostat]
The microtome knife needs to be chilled and maintained at low temperature to prevent ______
complete melting of the tissue
[cryostat]
When the tissue is too cold, on the other
hand, ______ is increased
resistance to cutting
The cryostat should be left on at all
times even when not in use, since it will ______
require several hours to reach
operating temperature from a room temperature start
It takes at least ______
for a knife to come down to operating temperature, so that a spare knife should always be kept inside the cryostat cabinet
one hour
What must be kept scrupulously clean and dry to ensure that the sections will cut smoothly and freely onto the knife surface? [cryostat]
Knife
Undersurface and edge of the anti-roll
May be used to clean the knife and anti-roll plate [cryostat]
Soft tissue paper (either dry or moistened with absolute alcohol)
The cryostat should be defrosted during the ___[1]___, including cleaning and oiling of microtome with ___[2]___
- weekend
- special low temp oil
[cryostat]
Require much lower temperatures to impart a suitable consistency for cutting
Fat or mucin
Hard or dense structures in a soft matrix
How to section:
Fat or mucin
Hard or dense structures in a soft matrix
Lowering the tissue or knife temperature or both
How to lower temperature of tissue/knife in cryostat for sectioning of:
Fat or mucin
Hard or dense structures in a soft matrix
Placing the block holder in a bath of alcohol or acetone containing dry ice,
Exposing the tissue to carbon dioxide
______ are generally used as mounting
media for tissue blocks that need to be sectioned on a cryostat
Synthetic water-soluble glycols and resins
Recommended mounting media for cryostat sectioned tissues
O.C.T. (Optimal Cutting Temperature) compound, Lab-Tek Products, Division of Miles Laboratories
Temperature of mounting media for cryostat
-5 to -15°C
-15 to -25°C
-35°C
Cryostat mounting media under -5 to -15°C
[BULL SSCT]
Brain
Uterine cuttering
Lymph nodes
Liver
Spleen
Soft cellular tumors
Cryostat mounting media under -15 to -25°C
[PNGOT]
Prostate
Non-fatty breast tissue
GI tract
Ovary
Tongue
Cryostat mounting media under 35°C
[FBOT]
Fatty breast
Omental tissue
[Cryostat]
Preferably, the tissue block
should be ___[1]___ mm. thick in order to ___[2]___
2-4
Minimize the risk of the knife hitting the metal tissue block holder
[cryostat]
Small fragments of tissue, such as curettings or brain biopsies, are placed on a ___[1]___
The blocks are then surrounded and covered with an ___[2]___, and frozen by ___[3]___
- thick base of O.C.T. compound
- additional matrix of O.C.T. compound
- liquid nitrogen
The frozen tissue is mounted on the microtome. Both the microtome knife
and the tissue block are left in the cryostat for ___[1]___ minutes at ___[2]___, to ensure that they are cooled to the correct temperature
- 15
- -20°C
[cryostat]
Sections between 5-10µm are
then cut ___[1]___, removed from the knife with a ___[2]___, attached directly to slides of cover-glasses at room temperature, air dried, and fixed (optional)
- slowly and steadily
- camel hair brush
To mount cryostat sections after cutting, one edge of the glass slide is
___[1]___
The section will ___[2]___
- lowered gently until it is about 1/2 to 1 mm. from the knife face
- automatically transfer from the cold knife to the relatively warm slide
[cryostat]
The slide should never be pressed down on the section, because this will cause a
___[1]___.
If this happens, ___[2]___
- frost mark to remain where the section rested on the knife
- the frost mark should be wiped away with soft tissue paper
Overall, cryostat sections provide the simplest, quickest and least labor intensive method for producing frozen sections, and are routinely used for ______ of surgical specimen
intraoperative and rapid diagnosis
It should be noted that cryostats cut only individual sections, and do not
______
form ribbons
Cryostat sections of fresh, unfixed tissue usually ___[1]___, and will preserve ___[2]___ that may be studied by histochemical techniques
- attach easily to the slide, even without adhesives
- enzymes and other substances
The cryostat is also recommended for any technique requiring cold
sectioning of fixed material, e.g., for ___[1]___, and for some special
methods for the ___[2]___
- fats and lipids
- nervous system
[Freezing Previously Fixed Tissue]
Sections of formalin-fixed tissue, however, may not adhere to the slide, and ______
will fall off or be detached during staining
[Freezing Previously Fixed Tissue]
Clean slides should be coated with ___[1]___ or ___[2]___ so that the fixed tissue will attach to the slide
- albumin
- chrome-glycerin jelly
Special fixatives such as ______ may be used in histochemistry and for lipid
demonstration
10% formol calcium at 4°C
[Freezing Previously Fixed Tissue]
Special fixatives such as 10% formol calcium at 4°C may be used in ______
histochemistry and for lipid
demonstration
Tissues that have been fixed or stored in alcohol should be ___[1]___ before sectioning, since ___[2]___
- washed in water for 12-24 hours
- alcohol inhibits freezing
[Freezing Previously Fixed Tissue]
Cumbersome way to attach fixed tissue to slide is to ______ before freezing and sectioning for rapid surgical diagnosis
immerse the tissue block in boiling 10% buffered formalin for 1 to 2 minutes
[nerve and muscle]
The portion of a specimen intended for frozen section should be transported on ___[1]___, and rapidly frozen within ___[2]___
- top of wet ice, on saline-dampened gauze
- two hours
