Infiltration, Embedding, Section-cutting /Sectioning, Mounting, Staining

Question 1

To completely displace the clearing agent, a typical paraffin infiltration sequence of paraffin for specimens not more than 4mm thick would be:

Answer 1

Paraffin wax 30 min
Paraffin wax 30 min
Paraffin wax 45 min

Question 2

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.

Answer 2

Infiltration

Question 3

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.

Answer 3

Infiltration

Question 4

Once the tissue has been processed it is ready to be oriented into a paraffin block and subsequently sectioned.

Answer 4

Embedding

Question 5

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.

Answer 5

Embedding

Question 6

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.

Answer 6

Embedding

Question 7

Correct orientation of tissue in a mold is the most important step in embedding.

Answer 7

Embedding

Question 8

Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.

Answer 8

Embedding

Question 9

Usually tissues are embedded with the surface to be cut facing down in the mold.

Answer 9

Embedding

Question 10

After orienting the section, the mold is filled with molten wax.

Answer 10

Embedding

Question 11

The main part of the labelled cassette is placed on top of the mold and topped up with more wax.

Answer 11

Embedding

Question 12

The whole mold is placed on a cold plate to solidify.

Answer 12

Embedding

Question 13

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.

Answer 13

Embedding

Question 14

The choice of mold will depend on the type of chuck in the microtome that will be used to section the tissue.

Answer 14

Embedding

Question 15

Stainless steel, ceramic, paper, plastic, and aluminum foil molds can be used.

Answer 15

Embedding

Question 16

The basic method is the same for each.

Answer 16

Embedding

Question 17

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.

Answer 17

Double embedding

Question 18

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.

Answer 18

Double embedding

Question 19

It also overcomes the difficulty of manipulating small tissue fragments during embedding and facilitates correct orientation and identification of tissues for histochemistry and immunohistochemistry.

Answer 19

Double embedding

Question 20

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.

Answer 20

Double embedding

Question 21

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.

Answer 21

Section-cutting

Question 22

Good microtomy techniques will minimize artifacts that can lead to difficult diagnostic interpretation of special stains.

Answer 22

Section-cutting

Question 23

One of the most directly correlated factors is the thickness in which a specimen is cut.

Question 24

Specimens for routine Hematoxylin and Eosin (H&E) are cut [?] in thickness.

Answer 24

3–5 μm

Question 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.

Answer 25

8–12 μm

2 μm

Question 26

does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy.

Answer 26

Paraffin wax

Question 27

[?] are the most commonly employed embedding media for semi-thin and ultrathin sections, but [?] are also used, particularly where immunohistochemistry is required.

Answer 27

Epoxy resins

acrylic resins

Question 28

Thicker sections (?) of resin-embedded tissue can also be cut for light microscopy.

Answer 28

0.35μm to 5μm

Question 29

Again, the immiscibility of most epoxy and acrylic resins with water necessitates the use of dehydration, usually with [?].

Answer 29

ethanol

Question 30

The [?] is nothing more than a knife with a mechanism for advancing a paraffin block across the knife.

Answer 30

microtome

Question 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 [?].

Answer 31

6 to 8 microns

Question 32

are sectioned with glass or diamond knives that can cut sections down to about 1 micron.

Answer 32

Plastic blocks (methacrylate, araldite, or epon)

Question 33

Thin sections for electron microscopy (0.25 micron) are best done with a

Answer 33

diamond knife.

Question 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

Answer 34

15 minutes

Question 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.

Answer 35

tearing, ripping, creases, holes or folding of sections

Question 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.

Answer 36

Section-cutting

Question 37

The tissue slide is drained and may be gently heated to evaporate the layer of water between the sections and the glass.

Answer 37

Section-cutting

Question 38

When all the water is gone, the slide may be heated enough to melt the wax, a procedure that may improve adhesion.

Answer 38

Section-cutting

Question 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.

Answer 39

Mounting

Question 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.

Answer 40

Mounting

Question 41

The temperature of the warm bath should be kept at 5–10oC below the melting point of the embedding wax.

Answer 41

Mounting

Question 42

If it is too hot, desiccated-looking sections will result, while cool water baths will produce excessive wrinkling of the tissue.

Answer 42

Mounting

Question 43

Adding a few drops of ethyl alcohol to the water may facilitate the mounting of tissue sections.

Answer 43

Mounting

Question 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.

Answer 44

Mounting

Question 45

Because tissue sections do not adhere well to untreated glass slides, a bonding agent also must be a component of the water bath.

Answer 45

Mounting

Question 46

Albumin, and poly-L-lysine are all suitable additives of this type.

Answer 46

Mounting

Question 47

One of the most serious issues faced in histotechnology is misidentification of tissues, which includes mislabeled specimens, block identification problems, and tissue contaminants.

Answer 47

Mounting

Question 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.

Answer 48

Mounting

Question 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.

Answer 49

floaters

Question 50

This may lead to an inaccurate diagnosis and result in medicolegal liabilities on both the pathologist and the histotechnologist.

Answer 50

floaters

Question 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.

Answer 51

Mounting

Question 52

Also, the technologist must routinely skim, or otherwise clear, the surface of the water bath between cases.

Answer 52

Mounting

Question 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.

Answer 53

tongue blade metastasis

Question 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.

Answer 54

Mounting

Question 55

The tissue sections mounted on the slide are nearly invisible under a light microscope so they must be stained to create contrast.

Answer 55

Staining

Question 56

After drying in a 60oC, the slide is passed through another series of chemical reagents.

Answer 56

Staining

Question 57

removes the paraffin and absolute alcohol removes the xylene.

Answer 57

Xylene

Question 58

The tissue on the slide is then rehydrated to prepare it for staining.

Answer 58

Staining

Question 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.

Answer 59

Staining

Question 60

The chemical properties of these dyes produce the visual appearance that is seen under the microscope.

Answer 60

Staining

Question 61

Different staining procedures are done depending on the tissue component that is being studied.

Answer 61

Staining

Question 62

Potential contamination during the staining procedure may be much higher than having “floaters” when mounting sections from a water bath.

Answer 62

Staining

Question 63

The tissue is deparaffinized during the first steps in preparing the slides for staining.

Answer 63

Staining

Question 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.

Answer 64

Staining

Question 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.

Answer 65

Staining

Question 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.

Answer 66

Staining

Question 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.

Answer 67

Staining

Question 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 (?).

Answer 68

“tissue processor”

Question 69

This machine allows the specimens to be infiltrated with a sequence of different solvents finishing in molten paraffin wax.

Answer 69

“tissue processor”

Question 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).

Answer 70

ethanol and xylene

Question 71

The duration and step details of the “processing schedule” chosen for a particular batch of specimens will depend on the [?] of the specimens.

Answer 71

nature and size

Question 72

The older design of an automatic issue processor is a [?] which contains a cage in which the tissue cassettes are placed.

Answer 72

carousel

Question 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.

Answer 73

carousel

Question 74

The [?] vertically agitates the cage in each solution before moving on to the next solution in the dehydration/ clearing method.

Question 75

The modern processors have a [?] in which the specimens are held and the different solutions are pumped in and out of the [?].

Answer 75

chamber

Question 76

In general, the whole process takes around [?] and is usually set up to run [?].

Answer 76

six hours

overnight

Question 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.

Answer 77

embedding

Question 78

Due to the viscosity of [?], some form of gentle agitation is highly desirable.

Answer 78

molten paraffin wax

Question 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.

Answer 79

ethanol

Question 80

If specimens are fresh they may incubate in [?] in the first stage on the machine.

Answer 80

formalin

Question 81

It is important to not keep the tissues in [?] too long or else they may become hard and brittle.

Answer 81

hot paraffin

Question 82

Processed tissues can be stored in the [?] at room temperature indefinitely.

Answer 82

cassettes

Question 83

is the impregnation of tissues by a molten medium under reduced pressure.

Answer 83

Vacuum infiltration

Question 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.

Answer 84

Vacuum infiltration

Question 85

Factors that impact the duration of tissue processing and extent of infiltration

Answer 85

Tissue Density and Thickness

Agitation

Temperature

Vacuum and pressure

Question 86

affects infiltration and subsequent sectioning of tissues.

Answer 86

Variable tissue density

Question 87

are usually more rapidly infiltrated than hard and dense tissues.

Answer 87

Spongy tissues (like lungs)

Question 88

also influences the rate of reagent diffusion and hence processing time.

Answer 88

Thickness of the tissue

Question 89

should be optimized for particular processing schedules, or alternatively, processing times should be adjusted to accommodate thick, thin or large tissue blocks.

Answer 89

Tissue thickness

Question 90

increases the flow of fresh fluids in and around the tissues.

Answer 90

Agitation using manual or automated processors

Question 91

Most tissue processing protocols utilize automated processors with [?] to speed fluid exchange.

Answer 91

vertical or rotary oscillation mechanisms

Question 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.

Answer 92

agitation

Question 93

Fluid interchange between processing reagents and tissues is promoted by exposure of the [?].

Answer 93

maximum tissue surface area

Question 94

Therefore, tissues should be [?] in baskets to facilitate exchange of reagents and increase diffusion.

Answer 94

loosely packed

Question 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.

Answer 95

perpendicular

Question 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.

Answer 96

37° to 45°C

Question 97

High temperature can cause the tissue [?], while low temperature [?] used in tissue processing, thereby reducing the rate of diffusion and increasing processing time.

Answer 97

to shrink and to become hard and brittle

increases the viscosity of reagents

Question 98

This can be avoided by maintaining embedding waxes [?].

Answer 98

2o to 3°C above their melting points

Question 99

Tissue shrinkage during infiltration in paraffin wax results mainly the effect of heat on [?].

Answer 99

collagen

Question 100

can increase the infiltration rate and decrease the time needed to complete steps in tissue processing protocols.

Answer 100

Reduced pressure

Question 101

facilitates infiltration of dense specimens with the more viscous embedding media.

Answer 101

High pressure

Question 102

[?] during tissue infiltration improves processing quality and can aid in removal of trapped air from porous tissue.

Answer 102

Vacuum application

Question 103

Using [?] during tissue processing protocols can reduce the infiltration time when dealing with dense and fatty tissue specimens.

Answer 103

Vacuum

Question 104

[?] applied during dehydration, clearing and infiltration improves the quality of processing in tissues such as lung which becomes de-aerated during the process.

Answer 104

Vacuum

Question 105

However, duration of wax infiltration is dependent upon [?] and is not generally reduced by applying a vacuum.

Answer 105

viscosity

Question 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.

Answer 106

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