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
Muscle and nerve biopsies are divided into separate portions that will allow for formalin fixation and paraffin embedding
unfixed snap-frozen for ___[1]___
fixation and resin embedding for ___[2]___ and in some rare cases for ___[3]___
- cryostat sections
- electron microscopy (EM)
- biochemical immunoblotting studies
[Examination of nerve and muscle]
Upon receipt in the histology lab, specimen is oriented in ___[1]___ compound and snap-frozen in liquid ___[2]___ for optimal results
- O.C.T. (Optimal Cutting Temperature)
- nitrogen/ isopentane
[Examination of nerve and muscle]
Are critical to obtaining undamaged sections of unfixed muscle fibers
Orientation, size, and expedient flash freezing
[Examination of nerve and muscle]
Do not allow the tissue to freeze slowly or to soak up excess saline, as these will cause:
artifacts that can be seen microscopically and can interfere with diagnostic interpretation
[Examination of nerve and muscle]
A portion of the tissue is oriented on a ___[1]___, fixed with ___[2]___ and processed for staining with ___[3]___
- piece of cardboard
- 10% buffered formalin
- routine Hematoxylin and Eosin (H&E staining)
[Examination of nerve and muscle]
The biopsy portion for electron microscopy is fixed in a ___[1]___ and postfixed in ___[2]___, usually by a ___[3]___
- buffered solution of glutaraldehyde
- osmium tetroxide
- specialist in electron microscopy
[Examination of nerve and muscle]
In some ______, biochemical techniques may also be required
muscular degenerative disorders
For histochemical evaluation involving enzyme studies, the tissue needs to
be ______, and the important chemical constituents should not have
been removed, altered or displaced
chemically active
Deemed to be the most ideal and preferred means of preserving tissues in order to avoid
complete or partial loss of enzymes consequent to chemical fixation
Frozen section
[SPECIAL PROCESSING TECHNIQUES]
Difficulties, however, arise in _______; since cut sections of tissue tend to disintegrate and cannot be easily handled without prior fixation
obtaining thin and serial sections of uniform thickness
In addition to fresh frozen tissue sectioning, there are methods that may be resorted to, if chemical fixation of tissue blocks is to be avoided, namely:
- Freeze-drying
- Freeze substitution
Like fresh frozen sections, special techniques have the common principle of rapidly preserving the tissue block by ______
freezing (quenching)
The aim of rapidly preserving tissue block by freezing is to ______ thereby preventing
chemical alteration of tissue and displacement of cellular tissue components
produce instant cessation of cellular activity
The aim of rapidly preserving tissue block by freezing is to produce instant cessation of cellular activity thereby ______
preventing chemical alteration of tissue and displacement of cellular tissue components
The use of isopentane, pentane and propane and most recently of dichloro-difluoromethane, which can be cooled to very low temperature in order to ______
retain the fluidity of the freezing agents
The use of ___[1-4]___, which can be cooled to very low temperature in order to retain the fluidity of the freezing agents
Isopentane,
Pentane
Propane
Dichloro-difluoromethane
A special way of preserving tissues by rapid freezing (quenching) of fresh tissue at -160°C and subsequently removing ice water molecules (dessication) by transferring the still frozen tissue block into a vacuum chamber at a higher temperature, e.g. -40°C (sublimation) without the use of any chemical fixative
Freeze-drying
Temp ng rapid freezing sa Freeze-Drying
-160degC
Temp ng vacuum chamber sa Freeze-Drying
-40degC
[freeze-drying]
A tissue around ___[1]___ thick is plunged into ___[2]___ mixture which has been chilled to -160° to -180°C with liquid nitrogen
- 2 mm.
- isopentane or propaneisopentane
[Freeze-Drying]
Tissues plunged into isopentane or propaneisopentane mixture which has been chilled to -160° to -180°C with liquid nitrogen will effectively solidify the tissue in _______
2-3 seconds
[Freeze-Drying]
Tissues plunged into isopentane or propaneisopentane mixture which has been chilled to -160° to -180°C with liquid nitrogen will effectively solidify the tissue in 2-3 seconds preventing ______
the formation of large ice crystals, autolysis and putrefaction
[Freeze-Drying]
Water is sublimated and dehydrated from the tissue, thereby completing the dessication process within _______.
24-48 hours
[Freeze-Drying]
Once drying is completed, the tissue is removed, fixed and embedded, either in ___[1]___, ___[2]___ or ___[3]___
- molten paraffin wax
- water soluble waxes
- celloidin
[Freeze-Drying]
Infiltration and impregnation are usually performed in a ______.
vacuum embedding oven
By far the most time consuming part of the Freeze-Drying process
Drying
Why drying is the most time-consuming procedure of freeze-drying?
certain tissues contain 70- 80% water by weight that has to be removed without damage to the tissue
Bakit mas mahirap i-section ang freeze-dried sections compared sa paraffin blocks?
The tissue is brittle and inadequately supported due to the relatively short period for wax impregnation
[Freeze-Drying]
The tissues are usually flattened directly into an ______ with the aid of the finger
albuminous glass slide
[Freeze-Drying]
___[1]___ must be avoided and ___[2]___ are preferred
- Water
- warm alcohol, acetone, mercury
Freeze-Drying produces ___[1]___tissue shrinkage, and allows tissues to be processed in a ___[2]___ state
- minimum
- fresh
Freeze-Drying causes minimal chemical change on the cells, most especially on the ___[1]___ components, and less ___[2]___
- protein
- displacement of tissue and cellular constituents
Freeze-drying may be used for special studies, including:
- Immunocytochemistry 2. Fluorescent antibody studies of polypeptide and polypeptide hormones
- Autoradiography
- Microspectrofluorimetry of autofluorescent substances
- Formaldehyde-induced fluorescence of biogenic amines (to demonstrate 5-hydroxytryptamine, adrenaline, and other catecholamines)
- Scanning electron microscopy
Freeze-drying demonstrates:
hydrolytic enzymes
mucous substances
glycogen
proteins
A process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration
Freeze-substitution
Difference of FREEZE-DRYING and FREEZE-SUBSTITUTION
FREEZE-DRYING
Dehydrated in a vacuum
FREEZE-SUBSTITUTION
Fixed in Rossman’s formula or in 1% Acetone
Dehydrated in absolute alcohol
Infiltration and embedding of Freeze-Substitution is then carried out in the same way as in ______
paraffin section
Freeze-substitution is based on rapid freezing of tissues followed by:
solution (“substitution”) of ice at temperatures well below 0°C
Freeze-substitution
A ___[size]___ specimen is thrown into ___[2]___ that is super cooled by liquid nitrogen to ___[3]___°C (with precautions)
size. 1-3 mm
- 3:1 propane - isopentane
- -175
[freeze-substitution]
Cryostat sections are cut ___[1]___ µm, and transferred to ___[2]___ (substituting fluid), and cooled to ___[3]___degC for ___[4]___ in order to dissolve ice slowly without distorting tissue structure
- 8-10
- water-free acetone
- -70
- 12 hours to 1 week
This technique is relatively more economical and less time-consuming than freeze-drying
Freeze-Substitution
For best morphological and histochemical preservation, substituting fluids should in general contain both ______
chemical fixing agent and solvent for ice
Freeze-Substitution substituting fluids:
1% osmium tetroxide in acetone
mercuric chloride in ethanol
picric acid in ethanol
Other name of infiltration
Impregnation
Other name of sectioning
Cutting / Microtomy
Describes the various steps required to take the tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome
Tissue processing
______ cannot be directly infiltrated with paraffin
Wet fixed tissues (in aqueous solutions)
Dehydration is usually done with increasing concentrations of alcohol _____
(70% to 95% to 100%)
Most common clearing agent
Xylene
Formalin, usually as a ______ solution, is the most popular fixative for preserving tissues that will be processed for paraffin embedding
phosphate-buffered
Specimens should remain in fixative long enough for it to ___[1]___ and then for an additional period in order to ___[2]___
- penetrate the tissue
- allow the chemical reactions of fixation to reach equilibrium (fixation time)
If fixation is not carried out under optimal conditions or if fixation is delayed, a tissue specimen can be:
irreversibly damaged
Formalin-fixed, paraffin-embedded tissues may be stored ______ at room temperature
indefinitely
Nucleic acids (both DNA and RNA) may be recovered from them ______ after fixation.
decades
Why is paraffin not miscible with water
Kasi hydrophobic
______ are removed at lower concentrations of ethanol [dehydration]
Water soluble proteins
When ethanol concentration is increased to 100% [dehydration], ______ may be dissolved
certain lipids
A typical dehydration sequence for specimens not more than 4mm thick would be:
70% ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min
Ethanol is a poor ___ solvent
fat
To ensure complete dehydration,
a superior fat solvent such as ___[1]___ should be added before the final absolute ethanol,
and ___[2]___ used as the transition solvent
- acetone or isopropanol
- chloroform or trichloroethane
[clearing]
The dehydrated tissue is transferred to an intermediate solvent that is fully miscible with ______
both ethanol and paraffin wax
The term “clearing” has been chosen because many (but not all) clearing agents impart an optical clarity or transparency to the tissue due to their ______
relatively high refractive index
Another important role of the clearing agent is to remove ______ from the tissue which otherwise presents a barrier to wax infiltration
a substantial amount of fat
A typical clearing sequence for specimens not more than 4mm thick would be:
Xylene 20 min
Xylene 20 min
Xylene 45 min
Paraffin wax is liquid at what temp
60degC
Paraffin wax is allowed to cool to ___°C in order to solidify into a consistency that allows sections to be cut
20
Paraffin wax are mixtures of purified paraffin wax and various additives that may include resins such as ______
styrene or polyethylene
A typical paraffin infiltration sequence of paraffin for specimens not more than 4mm thick would be:
Paraffin wax 30 min
Paraffin wax 30 min
Paraffin wax 45 min
The infiltrated tissue is removed from the cassette and very carefully oriented in a suitably sized metal mold so that the “______” can be determined
plane of section
Usually tissues are embedded with the surface to be cut facing ______
down in the mold
The choice of mold will depend on the ______ that will be used to section the tissue.
type of chuck in the microtome
The process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded
Double embedding
Double embedding is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as ______, then infiltrated a second time with wax in which they are also embedded
agar or nitrocellulose
Double embedding in agar-paraffin is a reliable and convenient method of handling minute and friable tissue fragments such as ______
curetting and endoscopic biopsies
The tissues may ______ by the time they are infiltrated with wax, but if adequately processed, they will still show ______ l and allow for accurate histopathological evaluation
- shrink
- good morphological detail
Specimens for routine Hematoxylin and Eosin (H&E) are cut _____ μm in thickness
3–5
Tissues to be examined for amyloid
deposits are better sectioned at _____ μm
8–12
Kidney biopsies should be cut
at _____ μm for optimal viewing of the structures of glomeruli
2
The most commonly employed embedding media for semi-thin and ultrathin sections
Epoxy resins
Acrylic resins are also used as embedding media, particularly where ______ is required
immunohistochemistry
Thicker sections (0.35μm to 5μm) of resin-embedded tissue can also be cut for ______
light microscopy
The immiscibility of most epoxy and acrylic resins with water necessitates the use of ______ , usually with ethanol.
dehydration
Knives are either of the standard ______ variety or ______ variety
thick metal
thin disposable
Usually this distance can be set, for most paraffin embedded tissues at ______ microns
6 to 8
Plastic blocks (methacrylate, araldite, or epon) are sectioned with ___ or _____ that can cut sections down to about ___ micron
glass
diamond knives
1
Example of plastic blocks
(methacrylate, araldite, or epon)
Thin sections for electron microscopy (0.25 micron) are best done with a ______ knife
diamond
The glass slides containing the specimen are then placed in a warm oven [after sectioning] for about ___ minutes to help the section adhere to the slide
15
If heat from oven (after microtomy) might harm such things as antigens for immunostaining, then this step can be bypassed and ______ can be used instead to pick up the sections
glue-coated slides
The temperature of the warm bath should be kept at ___ degC below the melting point of the embedding wax
5–10
If waterbath is too hot, ______ looking sections will result, while cool water baths will produce ______ of the tissue
desiccated-
excessive wrinkling
Adding a few drops of ______ to the water [mounting] may facilitate the mounting of tissue sections
ethyl alcohol
The ribbon must not be left in the bath for more than ______ minutes, or ______ of the tissue will be produced
1 or 2
over-hydration
Because tissue sections do not adhere well to untreated glass slides, a ______ also must be a component of the water bath. ______, and ______ are all suitable additives of this type
bonding agent
Albumin, poly-L-lysine
Tissue adheres to a wooden applicator stick that is used to float successively prepared ribbons from two different cases
tongue blade metastasis
In cases where the specimen is limited in size (such as ______, ______, ______), it is advisable to save any unmounted paraffin ribbon (with appropriate identification) from such cases for at least _____ after they are accessioned
- skin, bronchoscopy, gastrointestinal biopsies
- week
The tissue is ______ during the first steps in preparing the slides for staining
deparaffinized
Because the stainer baths are a potential reservoir of tissue contaminants, changing the staining fluids may alleviate some of the potential for ______ from this source
carryover
If the processor is to be run overnight, it should be programmed to hold on the ______ and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated
first ethanol bath
If specimens are fresh they may incubate in ______ in the first stage on the (automatic tissue processing) machine
formalin
It is important to not keep the tissues in hot paraffin too long or else they may become ______
hard and brittle
Processed tissues can be stored in the cassettes at room temperature ___[time period]___
indefinitely
Is the impregnation of tissues by a molten medium under reduced pressure
Vacuum infiltration
The procedure assists the complete and rapid impregnation of tissues with wax and reduces the time tissues are subjected to high temperatures, thereby minimizing heat-induced tissue hardening, facilitating complete removal of transition solvents, and prolonging the life of wax by reducing solvent contamination
Vacuum infiltration
Vacuum infiltration assists the complete and rapid impregnation of tissues with wax and reduces the time tissues are subjected to high temperatures, thereby:
minimizing heat-induced tissue hardening,
facilitating complete removal of transition solvents,
prolonging the life of wax by reducing solvent contamination
Factors that impact the duration of tissue processing and extent of infiltration
- Tissue Density and Thickness
- Agitation
- Temperature
- Vacuum and pressure
Agitation using manual or automated processors increases the ______ in and around the tissues
flow of fresh fluids
Most tissue processing protocols utilize automated processors with ______ oscillation mechanisms to speed fluid exchange
vertical or rotary
Without agitation, tissues tend to ______ or become ______, therefore reducing surface area available for fluid exchange
settle to the bottom of the processing device
too tightly packed
Fluid interchange between processing reagents and tissues is promoted by ______
exposure of the maximum tissue surface area
Tissues should be ______ to facilitate exchange of reagents and increase diffusion
loosely packed in baskets
Ideally, the cassette perforations should be ______ to the fluid flow
perpendicular
Temperatures in the range of ______°C, for a limited time can speed up fluid penetration and tissue processing protocols
37° to 45
High temperature can cause the tissue to _____ and to become _____
shrink
hard and brittle
Low temperature increases the ______ in tissue processing, thereby reducing the rate of diffusion
viscosity of reagents used
Tissue shrinkage during infiltration in paraffin wax results mainly the effect of heat on ______
collagen
Decreased rate of diffusion in automated tissue processing can be avoided by
Maintaining embedding waxes 2-3°C above their melting points
Reduced pressure can ______ the infiltration rate
increase
High pressure facilitates infiltration of dense specimens with the more ______ embedding media
viscous
______ during tissue infiltration improves processing quality and can aid in removal of trapped air from porous tissue
Vacuum application
Using vacuum during tissue processing protocols can ______ the infiltration time when dealing with dense and fatty tissue specimens
reduce
Using vacuum during tissue processing protocols can reduce the infiltration time when dealing with ______ tissue specimens
dense and fatty
Vacuum applied during dehydration, clearing and infiltration improves the quality of processing in tissues such as ______
lung which becomes de-aerated during the process
duration of wax infiltration is dependent upon ______ and is not generally reduced by applying a vacuum.
viscosity
______ and ______ will prevent the cells from detaching during staining
Gentle washing and minimal thickness of cell layers
In general, _____ and _____ should be incubated conservatively
needle biopsies and bloody specimens
______ specimens can be processed for longer than
average
fatty
