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HISTOPATHOLOGY > CHAPTER 11 IMPREGNATION AND EMBEDDING > Flashcards

CHAPTER 11 IMPREGNATION AND EMBEDDING Flashcards

1
Q

is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen.

A

Impregnation (Infiltration)

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2
Q

This allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.

A

Impregnation (Infiltration)

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3
Q

is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.

A

Embedding (Casting or Blocking)

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4
Q

The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes is known as an

A

Embedding Medium.

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5
Q

There are generally four types of impregnation and embedding medium, namely:

A
  1. Paraffin wax
  2. Celloidin (collodion)
  3. Gelatin
  4. Plastic
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6
Q

is the simplest, most common and best embedding medium used for routine tissue processing.

A

Paraffin

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7
Q

wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.

A

Paraffin

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8
Q

It is solid at room temperature but melts at temperatures up to about 65°C or 70°C.

A

Paraffin

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9
Q

can be purchased with melting points at different temperatures, the most common for histological use being about 56°C to 58°C.

A

Paraffin

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10
Q
  1. Thin individual serial sections may be cut with ease from the majority of tissues without distortion.
A

PARAFFIN WAX IMPREGNATION

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11
Q
  1. The process is very rapid, allowing sections to be prepared within 24 hours.
A

PARAFFIN WAX IMPREGNATION

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12
Q
  1. Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction.
A

PARAFFIN WAX IMPREGNATION

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13
Q
  1. Because formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, they are an important resource for historical studies in medicine.
A

PARAFFIN WAX IMPREGNATION

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14
Q
  1. Many staining procedures are permitted with good results.
A

PARAFFIN WAX IMPREGNATION

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15
Q
  1. Overheated paraffin makes the specimen brittle.
A

PARAFFIN WAX IMPREGNATION

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16
Q
  1. Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult.
A

PARAFFIN WAX IMPREGNATION

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17
Q
  1. Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken, and tissue blocks crumble when sectioned and break up when floated out in a water bath.
A

PARAFFIN WAX IMPREGNATION

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18
Q
  1. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support; otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable.
A

PARAFFIN WAX IMPREGNATION

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19
Q
  1. Paraffin processing is not recommended for fatty tissues. The dehydrating and clearing agents used in the process dissolve and remove fat from the tissues.
A

PARAFFIN WAX IMPREGNATION

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20
Q

The traditional advice with paraffin wax is to use this about [?] above its melting point.

A

2°C

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21
Q

depends upon the molecular weight of the components and the ambient temperature.

A

Wax hardness (viscosity)

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22
Q

To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above

A

60°C or 65°C.

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23
Q

Paraffin wax is traditionally marketed by its melting points which range from

A

39°C to 68°C.

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24
Q

depends upon the crystal morphology of the embedding medium.

A

Tissue-wax adhesion

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25
Q

After being completely cleared, the tissue is submerged in two or more changes of melted paraffin wax, either in a paraffin oven or in an incubator which has been regulated at

A

55-60°C.

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26
Q

The duration and number of changes required for thorough impregnation of tissue depends on:

A

Size and type of tissues: Longer time is required for thicker tissues.
Use of vacuum imbedding: Vacuum reduces the time required for complete impregnation.
Clearing agent employed

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27
Q

Common waxes have melting points of

A

45°C, 52°C, 56°C and 58°C.

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28
Q

The [?] is normally used for routine work. In a laboratory with temperature ranging from [?], paraffin wax with a melting point of [?] is indicated.

A

56°C wax

20-24°C

54-58°C

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29
Q

If the laboratory temperature is between [?], the melting point of wax to be used should be between [?].

A

15-18°C

50 and 54°C

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30
Q

There are three ways by which paraffin wax impregnation and embedding of tissues may be performed:

A
  1. By manual processing
  2. By automatic processing
  3. By vacuum embedding
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31
Q
  1. Manual Processing:
A

Fixation:
10% Buffered Formalin 24 hours

Dehydration:
70% Alcohol 6 hours
95% Alcohol 12 hours
100%Alcohol 2 hours
100% Alcohol 1 hour
100%Alcohol 1 hour

Clearing:
Xylene or Toluene 1 hour
Xylene or Toluene 1 hour

Impregnation:
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes

Embedding:
Paraffin wax 3 hours

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32
Q

At least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue.

A

Manual Processing

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33
Q

The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to insure complete embedding or casting of tissue.

A

Manual Processing

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34
Q

This method makes use of an automatic tissue processing machine (i.e., Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues. This results in a more rapid diagnosis with less technicality.

A

Automatic Processing

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35
Q

Usually, only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen. This is made possible due to constant tissue agitation which accelerates and improves tissue penetration giving rise to more consistent results.

A

Automatic Processing

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36
Q

The first [?] bath should be discarded, and the others moved down, so that the final bath has fresh [?] after two complete processing runs of loads of at least three-quarters capacity.

A

100% ethanol

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37
Q

The clearing agent and the dilute ethanols should be changed at least

A

once a week.

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38
Q

Wax bath thermostats should be set at least [?] above the melting point of the wax, and timing should be checked when loading the machine, especially if the machine is equipped with a delay mechanism.

A

3 degrees

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39
Q

involves wax impregnation under negative atmospheric pressure inside an embedding oven.

A

Vacuum Embedding

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40
Q

involves wax impregnation under negative atmospheric pressure inside an embedding oven.

A

Vacuum Embedding

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41
Q

It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening.

A

Vacuum Embedding

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42
Q

It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination.

A

Vacuum Embedding

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43
Q

hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of thetissue.

A

Vacuum

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44
Q

This technique is particularly recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system.

A

Vacuum Embedding

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45
Q

requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 760 mm Hg is applied using a water or mechanical pump.

A

Vacuum infiltration

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46
Q

Of the three methods of [?] gives the fastest result.

A

paraffin wax impregnation, vacuum impregnation

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47
Q

are easily removed from the tissues

A

Benzene and xylene

48
Q

are more difficult to remove and require more frequent wax changes.

A

chloroform and cedarwood oil

49
Q

Addition of [?] may hasten displacement of cedarwood oil with less tissue shrinkage.

A

benzene

50
Q

Since prolonged treatment in melted paraffin causes shrinkage and hardening of tissues, making cutting difficult, the tissue should not be left in the paraffin oven for more than

A

4 hours.

51
Q

Tissues become increasingly harder and more brittle as they are heated. Infiltration in overheated paraffin (above [?]) will also produce shrinkage and hardening of tissues and destroy lymphoid tissues completely. To avoid this, the paraffin oven must be maintained at a temperature [?] above the melting point of paraffin to be used for impregnation.

A

60°C

2 to 5°C

52
Q

Fresh wax should be filtered before use in a wax oven at a temperature [?] higher than its melting point.

A

2°C

53
Q

Water must therefore be removed by heating the wax to [?], thereby raising its melting point.

A

100 -105°C

54
Q

SUBSTITUTES FOR PARAFFIN WAX:

A
55
Q

is a mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C.

A

Paraplast

56
Q

It is more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding.

A

Paraplast

57
Q

Blocks obtained are more uniform than with any other medium, with better ribboning of sections.

A

Paraplast

58
Q

Serial sections may be cut with ease, without cooling the tissue block, thereby preventing the formation of ice crystal artefacts.

A

Paraplast

59
Q

No deposit is left on the slide after staining, and no special processing schedule is required.

A

Paraplast

60
Q

It is soluble in common clearing agents and follows the same time schedule for paraffin impregnation, and does not tend to crack like other paraffin wax substitutes.

A

Paraplast

61
Q

Generally, [?] with a melting point of 56 to 58oC is recommended.

A

Paraplast

62
Q

During the winter, 54 to 56oC Paraplast may be used if the tissue is cut in a cool room. During the summer it may be necessary to use 60 to 63oC, although this is to be avoided if possible in order to not to “cook” the tissue. “Cooked” tissue does not section well or, if it does, it does not stain well and most details are destroyed.

A

Paraplast

63
Q

is synthetic wax substitute similar to Paraplast with a melting point of 56-58°C.

A

Embeddol

64
Q

It is less brittle and less compressible than Paraplast.

A

Embeddol

65
Q

is a semisynthetic wax recommended for embedding eyes.

A

Bio/aid

66
Q

is a product of paraffin, containing rubber, with the same property as Paraplast.

A

Tissue Mat

67
Q

has a lower melting point (46-48°C), but it is harder than paraffin.

A

Ester Wax

68
Q

It is not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents; hence, it can be used for impregnation without prior clearing of the tissue.

A

Ester Wax

69
Q

may be used as clearing agents, if indicated.

A

Cellosolve (ethylene glycol monoethyl ether) or xylene

70
Q

are plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C.

A

Water Soluble Waxes

71
Q

are incorporated in the majority of proprietary histological paraffin wax blends presently available to improve adhesion, hardness and plasticity.

A

Polymer waxes

72
Q

The most commonly used Polymer wax is [?], a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature.

A

Carbowax

73
Q

It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue.

A

Carbowax

74
Q

added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

A

Dimethyl sulphoxide (DMSO)

75
Q

scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.

A

DMSO

76
Q

is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo.

A

Celloidin (Collodion)

77
Q

It is supplied in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol.

A

Celloidin (Collodion)

78
Q

This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain.

A

Celloidin (Collodion)

79
Q

No heat is required, and the resultant block has a rubbery consistency which gives good support to the tissues.

A

Celloidin (Collodion)

80
Q

Disadvantages include inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take several weeks or months).

A

Celloidin (Collodion)

81
Q

Two methods are used for celloidin impregnation of tissue:

A

Wet Celloidin Method

Dry Celloidin Method

82
Q
  • is recommended for bones, teeth, large brain sections and whole organs.
A

Wet Celloidin Method

83
Q

is preferred for processing of whole eye sections.

A

Dry Celloidin Method

84
Q

is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly. Because of this, many workers prefer L.V.N. to the ordinary celloidin for impregnation and embedding.

A

Low Viscosity Nitrocellulose (L.V.N.)

85
Q

It forms a harder tissue block and makes cutting of thinner sections possible.

A

Low Viscosity Nitrocellulose (L.V.N.)

86
Q

The tendency of tissues to crack may be prevented by adding plasticizers (e.g. oleum ricini or castor oil) when embedding chrome-mordanted tissues.

A

Low Viscosity Nitrocellulose (L.V.N.)

87
Q

Paraffin embedded tissues are arranged at the bottom of the mold together with their proper labels and immersed in melted paraffin at a temperature between [?] above its melting point and then cooled rapidly in a refrigerator at [?] or immersed in cold water to solidify.

A

5-10°C

-5°C

88
Q

The process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining, is known as

A

Orientation.

89
Q

Several types of Blocking-out Molds may be used:

A
  1. Leuckhart’s Embedding Mold
  2. Compound Embedding Unit
  3. Plastic Embedding Rings and Base Mold
  4. Disposable Embedding Molds
90
Q
  • consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen .
A

Leuckhart’s Embedding Mold

91
Q

It is recommended for routine use, although, too slow and cumbersome for use in a busy laboratory.

A

Leuckhart’s Embedding Mold

92
Q

is made up of a series of interlocking plates resting on a flat metal base, forming several compartments.

A

Compound Embedding Unit

93
Q

-consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.

A

Plastic Embedding Rings and Base Mold

94
Q

disposable thin plastic embedding molds, available in 3 different sizes, are simply peeled off one at a time, as soon as the wax has solidified, giving perfect even block without trimming.

A

Peel-Away

95
Q

It may be placed directly in the chuck or block holder of the microtome.

A

Peel-Away

96
Q

-such as those used in ordinary refrigerators may be recommended for busy routine laboratories.

A

Plastic Ice Trays

97
Q

are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.

A

Paper Boats

98
Q

They have the advantage of being cheap and easy to make.

A

Paper Boats

99
Q

They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks.

A

Paper Boats

100
Q

used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used.

A

Celloidin or Nitrocellulose Embedding Method

101
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 paraffin wax in which they are subsequently embedded.

A

Double-Embedding

102
Q

This is used to facilitate cutting of large blocks of dense firm tissues like the brain.

A

Double-Embedding

103
Q

They are also recommended for making small sections of celloidin blocks.

A

Double-Embedding

104
Q

The introduction of plastic resin embedding media has provided superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for high resolution light microscopy of tissue sections thinner than the usual [?], such as renal biopsies andbone marrow biopsies.

A

4-6 μm

105
Q

Plastics are classified into [?], based on their chemical composition.

A

epoxy, polyester, or acrylic

106
Q

are made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators.

A

Epoxy embedding plastics

107
Q

Three types of epoxy plastics are used in microscopy, i.e., those based on either bisphenol A (?), or glycerol (?), or cyclohexene dioxide (?).

A

Araldite

Epon

Spurr

108
Q

Infiltration by [?] is slow, partly because the epoxy plastic itself is a large molecule.

A

Araldite

109
Q

have a lower viscosity but are often sold as mixtures of isomers.

A

glycerol-based epoxy plastics (Epon)

110
Q

can be obtained pure, have very low viscosity, and infiltrate fastest.

A

Cyclohexene dioxide-based plastics (Spurr)

111
Q

were originally introduced for electron microscopy in the mid- 1950s, but have been superseded by more superior epoxides, and are now seldom used.

A

Polyester plastics

112
Q

are made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy.

A

Acrylic plastics

113
Q

has proved to be a popular embedding medium for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes.

A

Polyglycol methacrylate (GMA)

114
Q

The polar water soluble, 2-hydroxyethyl methacrylate, commonly known as [?], has found an increasing number of applications for the embedding of biological tissue for transmission electron microscopy (TEM), for the preservation and observation of fine structure not previously subjected to solvent dehydration.

A

“glycol methacrylate”, or GMA

115
Q

forms only non-crosslinked straight chains on polymerization and therefore requires no hardener.

A

GMA

116
Q

is added to the plastic as a catalyst that decomposes to form phenyl radicals acting as an active site for the polymerization of acrylics.

A

Benzoyl peroxide

117
Q

Acrylic plastics based on [?] are also widely used because of its hardness as the ideal embedding medium for undecalcified bone and is widely used for bone histomorphometry and bone marrow hematopathology.

A

methyl methacrylate (MMA)

HISTOPATHOLOGY (14 decks)

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