Infiltration, Embedding, Section-cutting /Sectioning, Mounting, Staining Flashcards
1
Q
To completely displace the clearing agent, a typical paraffin infiltration sequence of paraffin for specimens not more than 4mm thick would be:
A
Paraffin wax 30 min
Paraffin wax 30 min
Paraffin wax 45 min
2
Q
The cleared tissue is infiltrated with a suitable histological wax (usually paraffin) which is liquid at 60°C and then allowed to cool to 20°C in order to solidify into a consistency that allows sections to be cut.
A
Infiltration
3
Q
These waxes are mixtures of purified paraffin wax and various additives that may include resins such as styrene or polyethylene that allow them to be sectioned thin enough on a microtome, forming ribbons that can flatten fully when floated on a warm water bath.
A
Infiltration
4
Q
Once the tissue has been processed it is ready to be oriented into a paraffin block and subsequently sectioned.
A
Embedding
5
Q
After infiltration with wax, the tissue is oriented and placed in a mold that is filled with molten wax to form a solid tissue block that can later be clamped into a microtome for sectioning.
A
Embedding
6
Q
The infiltrated tissue is removed from the cassette and very carefully oriented in a suitably sized metal mold so that the “plane of section” can be determined.
A
Embedding
7
Q
Correct orientation of tissue in a mold is the most important step in embedding.
A
Embedding
8
Q
Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.
A
Embedding
9
Q
Usually tissues are embedded with the surface to be cut facing down in the mold.
A
Embedding
10
Q
After orienting the section, the mold is filled with molten wax.
A
Embedding
11
Q
The main part of the labelled cassette is placed on top of the mold and topped up with more wax.
A
Embedding
12
Q
The whole mold is placed on a cold plate to solidify.
A
Embedding
13
Q
When this is completed the block with its attached cassette can be removed from the mold and is ready to be sectioned on a microtome.
A
Embedding
14
Q
The choice of mold will depend on the type of chuck in the microtome that will be used to section the tissue.
A
Embedding
15
Q
Stainless steel, ceramic, paper, plastic, and aluminum foil molds can be used.
A
Embedding
16
Q
The basic method is the same for each.
A
Embedding
17
Q
is 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.
A
Double embedding
18
Q
in agar-paraffin is a reliable and convenient method of handling minute and friable tissue fragments such as curetting and endoscopic biopsies, which can be lost during tissue processing.
A
Double embedding
19
Q
It also overcomes the difficulty of manipulating small tissue fragments during embedding and facilitates correct orientation and identification of tissues for histochemistry and immunohistochemistry.
A
Double embedding
20
Q
The tissues may shrink by the time they are infiltrated with wax, but if adequately processed, they will still show good morphological detail and allow for accurate histopathological evaluation.
A
Double embedding
21
Q
Once the tissues have been embedded, they must be cut into sections that are thin enough to be placed on a slide. This is done with a microtome.
A
Section-cutting
22
Q
Good microtomy techniques will minimize artifacts that can lead to difficult diagnostic interpretation of special stains.
A
Section-cutting
23
Q
One of the most directly correlated factors is the thickness in which a specimen is cut.
A
24
Q
Specimens for routine Hematoxylin and Eosin (H&E) are cut [?] in thickness.
A
3–5 μm
25
Tissues to be examined for amyloid deposits are better sectioned at [?], whereas kidney biopsies should be cut at [?] for optimal viewing of the structures of glomeruli.
8–12 μm
2 μm
26
does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy.
Paraffin wax
27
[?] are the most commonly employed embedding media for semi-thin and ultrathin sections, but [?] are also used, particularly where immunohistochemistry is required.
Epoxy resins
acrylic resins
28
Thicker sections (?) of resin-embedded tissue can also be cut for light microscopy.
0.35μm to 5μm
29
Again, the immiscibility of most epoxy and acrylic resins with water necessitates the use of dehydration, usually with [?].
ethanol
30
The [?] is nothing more than a knife with a mechanism for advancing a paraffin block across the knife.
microtome
31
Knives are either of the standard thick metal variety or thin disposable variety (like a disposable razor blade). Usually this distance can be set, for most paraffin embedded tissues at [?].
6 to 8 microns
32
are sectioned with glass or diamond knives that can cut sections down to about 1 micron.
Plastic blocks (methacrylate, araldite, or epon)
33
Thin sections for electron microscopy (0.25 micron) are best done with a
diamond knife.
34
The glass slides containing the specimen are then placed in a warm oven for about [?] to help the section adhere to the slide. If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue- coated slides can be used instead to pick up the sections. It
15 minutes
35
It is important to have a properly fixed and embedded block or much artifact can be introduced during the sectioning. Common artifacts include [?], etc.
tearing, ripping, creases, holes or folding of sections
36
Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide.
Section-cutting
37
The tissue slide is drained and may be gently heated to evaporate the layer of water between the sections and the glass.
Section-cutting
38
When all the water is gone, the slide may be heated enough to melt the wax, a procedure that may improve adhesion.
Section-cutting
39
Paraffinized ribbons of serial tissue sections can be removed from the microtome knife as they are cut, by using a wooden tongue depressor blade.
Mounting
40
In this process, a slight traction is exerted on the end of the ribbon, stretching it gradually over the wooden blade while floating in a warm water bath.
Mounting
41
The temperature of the warm bath should be kept at 5–10oC below the melting point of the embedding wax.
Mounting
42
If it is too hot, desiccated-looking sections will result, while cool water baths will produce excessive wrinkling of the tissue.
Mounting
43
Adding a few drops of ethyl alcohol to the water may facilitate the mounting of tissue sections.
Mounting
44
The ribbon must not be left in the bath for more than 1 or 2 minutes, or over-hydration of the tissue will be produced, simulating the appearance of edema fluid when examined microscopically.
Mounting
45
Because tissue sections do not adhere well to untreated glass slides, a bonding agent also must be a component of the water bath.
Mounting
46
Albumin, and poly-L-lysine are all suitable additives of this type.
Mounting
47
One of the most serious issues faced in histotechnology is misidentification of tissues, which includes mislabeled specimens, block identification problems, and tissue contaminants.
Mounting
48
A potentially dangerous mistake that can take place when mounting sections from flotation baths is the “shedding” of friable small tissue fragments that float freely on the surface of the water, and which may be inadvertently picked up when mounting slides from subsequently processed but unrelated cases.
Mounting
49
Known as ‘‘?’’, these tiny pieces of unrelated tissue commonly cause problems in interpretation of the biopsies by the pathologist. For example, a small piece of unrelated cancerous tissue may inadvertently “float” and be deposited on the slides of a subsequent case that is being evaluated for the presence of malignancy.
floaters
50
This may lead to an inaccurate diagnosis and result in medicolegal liabilities on both the pathologist and the histotechnologist.
floaters
51
Meticulous cleaning of the microtome water bath and frequent clearing or changing of the water will alleviate the danger of rare contaminants being carried over to subsequent sections being mounted.
Mounting
52
Also, the technologist must routinely skim, or otherwise clear, the surface of the water bath between cases.
Mounting
53
Another source of floater-type artefact is the ‘‘?,’’ wherein tissue adheres to a wooden applicator stick that is used to float successively prepared ribbons from two different cases.
tongue blade metastasis
54
In cases where the specimen is limited in size (such as skin, bronchoscopy and gastrointestinal biopsies), it is advisable to save any unmounted paraffin ribbon (with appropriate identification) from such cases for at least a week after they are accessioned, so that remounts can be prepared when additional sections are requested, without the need for further microtomy of the tissue block.
Mounting
55
The tissue sections mounted on the slide are nearly invisible under a light microscope so they must be stained to create contrast.
Staining
56
After drying in a 60oC, the slide is passed through another series of chemical reagents.
Staining
57
removes the paraffin and absolute alcohol removes the xylene.
Xylene
58
The tissue on the slide is then rehydrated to prepare it for staining.
Staining
59
Most staining procedures in the laboratory, aside from antibody-based immunohistochemistry (IHC), use chemicals or dyes that will bind or have affinity for certain components of the cells and extracellular components.
Staining
60
The chemical properties of these dyes produce the visual appearance that is seen under the microscope.
Staining
61
Different staining procedures are done depending on the tissue component that is being studied.
Staining
62
Potential contamination during the staining procedure may be much higher than having “floaters” when mounting sections from a water bath.
Staining
63
The tissue is deparaffinized during the first steps in preparing the slides for staining.
Staining
64
As the slides are dipped up and down into the staining baths, the deparaffinized tissue can fragment and small dis-cohesive pieces can break free and be lifted on the slide.
Staining
65
Contamination in the staining solution is dependent on the volume of slides being stained and the time point during the day that the samples are taken.
Staining
66
Because the stainer baths are a potential reservoir of tissue contaminants, changing the staining fluids may alleviate some of the potential for carryover from this source.
Staining
67
And, as higher numbers of the contaminating fragments are localized to the first xylenes and alcohols, frequently changing these baths in particular may also be useful.
Staining
68
Tissue processing can be performed manually (hand processing), but when there is a large volume of tissues that need to be processed, it is more convenient and much more efficient to use an automated tissue processing machine (?).
“tissue processor”
69
This machine allows the specimens to be infiltrated with a sequence of different solvents finishing in molten paraffin wax.
“tissue processor”
70
The specimens are in an aqueous environment to start with (water-based) and must be passed through multiple changes of dehydrating and clearing solvents (typically [?]) before they can be placed in molten wax (which is hydrophobic and immiscible with water).
ethanol and xylene
71
The duration and step details of the “processing schedule” chosen for a particular batch of specimens will depend on the [?] of the specimens.
nature and size
72
The older design of an automatic issue processor is a [?] which contains a cage in which the tissue cassettes are placed.
carousel
73
This [?] has a number of glass beakers containing solvents and solutions which ensure that the tissue is dehydrated and cleared ready for paraffin wax embedding.
carousel
74
The [?] vertically agitates the cage in each solution before moving on to the next solution in the dehydration/ clearing method.
75
The modern processors have a [?] in which the specimens are held and the different solutions are pumped in and out of the [?].
chamber
76
In general, the whole process takes around [?] and is usually set up to run [?].
six hours
overnight
77
Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them. This "?" process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin.
embedding
78
Due to the viscosity of [?], some form of gentle agitation is highly desirable.
molten paraffin wax
79
If the processor is to be run overnight, it should be programmed to hold on the first [?] bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated.
ethanol
80
If specimens are fresh they may incubate in [?] in the first stage on the machine.
formalin
81
It is important to not keep the tissues in [?] too long or else they may become hard and brittle.
hot paraffin
82
Processed tissues can be stored in the [?] at room temperature indefinitely.
cassettes
83
is the impregnation of tissues by a molten medium under reduced pressure.
Vacuum infiltration
84
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
85
Factors that impact the duration of tissue processing and extent of infiltration
Tissue Density and Thickness
Agitation
Temperature
Vacuum and pressure
86
affects infiltration and subsequent sectioning of tissues.
Variable tissue density
87
are usually more rapidly infiltrated than hard and dense tissues.
Spongy tissues (like lungs)
88
also influences the rate of reagent diffusion and hence processing time.
Thickness of the tissue
89
should be optimized for particular processing schedules, or alternatively, processing times should be adjusted to accommodate thick, thin or large tissue blocks.
Tissue thickness
90
increases the flow of fresh fluids in and around the tissues.
Agitation using manual or automated processors
91
Most tissue processing protocols utilize automated processors with [?] to speed fluid exchange.
vertical or rotary oscillation mechanisms
92
Without [?], tissues tend to settle to the bottom of the processing device or become too tightly packed, therefore reducing surface area available for fluid exchange.
agitation
93
Fluid interchange between processing reagents and tissues is promoted by exposure of the [?].
maximum tissue surface area
94
Therefore, tissues should be [?] in baskets to facilitate exchange of reagents and increase diffusion.
loosely packed
95
Ideally, the cassette perforations should be [?] to the fluid flow. If tissues are allowed to settle on the bottom of a container or are too tightly packed, tissue surface area available for fluid exchange will be severely restricted.
perpendicular
96
Temperatures in the range of [?], for a limited time can speed up fluid penetration and tissue processing protocols. However, heat must be carefully monitored.
37° to 45°C
97
High temperature can cause the tissue [?], while low temperature [?] used in tissue processing, thereby reducing the rate of diffusion and increasing processing time.
to shrink and to become hard and brittle
increases the viscosity of reagents
98
This can be avoided by maintaining embedding waxes [?].
2o to 3°C above their melting points
99
Tissue shrinkage during infiltration in paraffin wax results mainly the effect of heat on [?].
collagen
100
can increase the infiltration rate and decrease the time needed to complete steps in tissue processing protocols.
Reduced pressure
101
facilitates infiltration of dense specimens with the more viscous embedding media.
High pressure
102
[?] during tissue infiltration improves processing quality and can aid in removal of trapped air from porous tissue.
Vacuum application
103
Using [?] during tissue processing protocols can reduce the infiltration time when dealing with dense and fatty tissue specimens.
Vacuum
104
[?] applied during dehydration, clearing and infiltration improves the quality of processing in tissues such as lung which becomes de-aerated during the process.
Vacuum
105
However, duration of wax infiltration is dependent upon [?] and is not generally reduced by applying a vacuum.
viscosity
106
Technical Considerations
-Baskets and metal cassettes should be [?].
-Tissues should not be [?] in baskets so as to impede fluid exchange.
-Processors must be free of [?].
-Fluid levels must be [?] than the specimen containers.
-Timing and delay mechanism must be [?]against the appropriate processing schedule.
-A processor log should be kept in which the number of specimens processed, processing reagent changes, temperature checks on the wax baths and the completion of the routine maintenance schedule are recorded as part of [?].
-[?] of cell layers will prevent the cells from detaching during staining.
-Make sure that there are [?] to make a diagnosis, and ensure that the reagents have been applied evenly to the slides.
-Quality of staining can be compromised by [?] and similarly by poor tissue processing.
-A good technician must evaluate and determine the processing of choice for each purpose, i.e., [?].
-Make sure glass slides are [?].
-In general, needle biopsies and bloody specimens should be [?], whereas fatty specimens can be processed for longer than average.
clean and wax-free
packed too tightly
spilled fluids and accumulated wax accumulations
higher
correctly set and checked
quality assurance program
Gentle washing and minimal thickness
enough sections
inadequate fixation
special stains on paraffin, frozen or cell smear preparations
clean and free from debris
incubated conservatively
