HISTOPATH UNIT 7-10 Flashcards
1
Q
Cellosolve is used as an embedding medium.
A
False (Cellosolve is a dehydrating agent)
2
Q
Test tubes is one of the possible embedding
molds.
A
TRUE
small fragments
3
Q
After embedding, 10 ̊C temperature prevents
cracking of the tissue block
A
TRUE
4
Q
Process of saturating the tissue with a medium,
usually liquid paraffin, to permeate or fill up the natural
cavities, spaces, and interstices of the tissue
A
IMPREGNATION
5
Q
A suitable embedding mold is filled with the molten
wax, the tissue is placed in it and oriented so it is
sectioned in the proper plane.
A
embedding
6
Q
Wax to be used must contain no trace of
clearing agent, dust particles, and must be
rapidly cooled to reduce the wax crystal
size.
A
TRUE
7
Q
A variety of molds can be used depending on the
technician’s preference.
A
TRUE
8
Q
Pertains to how you would like the tissue to
appear in the slide.
A
Orientation
9
Q
The tissue is oriented below the block.
■ Cut at the bottom: tissue block will
be at the front and the block will be
at the back.
A
TRUE
10
Q
When dealing with tissue blocks, always
orient the
A
wider side facing down
11
Q
There are two sides: select the side
with the most significant
pathologic findings.
A
TRUE
12
Q
For hollow organs (like the intestine,
fallopian tube, aorta, uterus, or vena cava,
etc), orient the tissue so that it will be cut in
A
cross section
13
Q
Take Note: tips of forceps are heated in an
alcohol lamp or in a forceps warmer, tips
should be hot enough so paraffin does not
solidify, but not so hot as to cause paraffin to
smoke.
A
TRUE
14
Q
Fill the bottom of the mold with a small amount of
paraffin. The depth of the mold should be at least
A
TWICE THE THICKNESS
15
Q
Pick up tissue, and place into the mold. Manipulation of the tissue in the mold must be quick, so paraffin does not begin to harden
A
TRUE
16
Q
After tissue is in the mold, fill the mold entirely with the paraffin. As the paraffin begins to harden, insert a code number label; the label should not go down to the bottom of the paraffin.
A
TRUE
17
Q
Allow the surface of the paraffin block to harden, then immerse the mold into a shallow, cool(______ ̊ C) water bath for about _______to hasten the solidification of the paraffin
A
10 degrees
10-15 minutes
18
Q
temp to prevent cracking of the tissue
block
A
10
19
Q
The paraffin will appear clear and homogeneous and there is no layering of the paraffin. Paraffin
demonstrating these conditions is best for sectioning.
A
TRUE
CLEAR AND HOMOGENOUS
20
Q
Rapidly converted from solid to liquid form on heating.
● Permeates the tissue in a liquid state.
● Solidifies relatively quickly on cooling.
A
PARAFFIN
21
Q
Becomes fluid on heating to a temperature which will not damage the tissue.
● When the paraffin solidifies it becomes firm enough to section at room temperature.
A
paraffin
22
Q
melting point
A
55 degrees
23
Q
Time of infiltration and subsequent embedding are
relatively short for small pieces of tissue.
★ Thin sections can be cut with the rotary microtome and sections will adhere to each other to form a ribbon.
★ Tissue once infiltrated and embedded can be stored in a dry condition indefinitely without damage to the
tissue
A
advantages of paraffin
24
Q
Mixture of highly purified paraffin and
synthetic plastic polymers.
SUBSTITUTE OF PARAFFIN WAX
A
paraplast
25
It is less brittle and less compressible than
Paraplast. It is a semisynthetic wax
recommended for embedding eyes.
embeddol
26
Has a lower melting point(46-48°C), but it is
harder than paraffin.
It is not soluble in water, but is soluble in
95% Ethyl Alcohol and other clearing
agents
ester wax (46-48)
27
With melting points of 38-42°C or 45-56°C
○ A polyethylene glycol, is suitable for many
enzyme histochemical studies.
○ Cytologic details are excellently preserved.
water soluble waxes
38-42
45-46
28
Histotechnologists may experience an
unpleasant and annoying oyster or garlic
taste.
DMSO
29
Elastic and resilient.
Highly purified paraffin waxes with DMSO
(dimethylsulfoxide)
30
Distortion of the histology of the tissue due to
shrinkage may occur, especially when sections are
being attached to glass slides (paraffin artifact).
☹ Sectioning of paraffin is difficult at high temperatures.
☹ Time for infiltration of large blocks of tissue is
excessive.
☹ Attachments of paraffin to the tissue.
DISADAVNTAGES OF PARAFFIN
31
Amorphous, slightly yellowish substance.
● Purified form of collodion or nitro-cellulose.
● For hard tissue specimens.
● For bigger cuts.
● 2 methods:
CELLOIDIN: WET CELLOIDIN AND DRY
32
Does not require heat;
★ Has a rubbery consistency;
★ Minimal distortion of specimen.
advantages of celloidin
33
Difficult to cut thin sections;
☹ Serial sections are difficult to prepare;
☹ Slow process
☹ Blocks and sections must be stored in 70% alcohol
otherwise they become discolored, dry, and shrunken.
disadvantages of celloidin
34
Low viscosity; allows higher concentration to be
used.
★ Greater speed of impregnation.
★ Final block is harder, allowing thinner sections to be cut.
★ Has a greater water tolerance than celloidin.
LVN
35
Have a tendency to crack down during handling and
staining.
● Take Note: use 0.5% oleum ricini (castor oil)
to minimize this tendency.
Envelopes tissues and prevents it
from breaking.
☹ Highly explosive.
DISADVANTAGES OF LVN
USE 0.5% oleum ricini castor oil
36
Tissue is first impregnated with celloidin, and
subsequently blocked in paraffin wax.
double embedding
37
Used in dealing with hard tissues.
○ Hard to ribbon, usually becomes brittle.
● For maintenance of the morphological appearance of the tissue.
● Serial sections are easily prepared.
● Extra degree of resilience is given when cutting hard
tissues.
double embedding
38
Main use is in double embedding technique with ester wax or paraffin wax
AGAR
39
Cohesive agent for multiple fragments or friable tissue
● Also an embedding medium, but the consistency is harder = higher ratio → more agar, less water.
AGAR
40
Has a lower melting point than agar.
● Main use in the production of whole organ sections
● For friable tissues
gelatin
41
Tissue can be embedded directly from water.
● However, it is restricted, due to the violent diffusion
currents which can lead to the complete
fragmentation of the section.
water soluble waxes
42
EPOXY
POLYESTER
ACRYLIC
plastic embedding medium
43
Reduces antigenicity, toxic, and
damages tissue.
● Examples:
○ Bisphenol A(Araldite);
○ Glycerol(Epon);
○ Cyclohexene dioxide(Spurr).
EPOXY
44
Not often used in plastic embedding mediums
polyester
45
Used extensively for light
microscopy.
● MMA
● GMA
acrylic
46
are used to provide shape and contain the
embedding mediums and tissue, allowing it to form upon
cooling.
molds
47
Molds for routine work and are widely used.
● Consist of 2 L-shaped pieces of metal.
● Arranged on a glass metal plate to form a mold of
desired size.
leuckharts embedding mold
48
One of the oldest. Size is dependent on orientation or
the distance of one from the other.
● One of the reusable molds.
leuckharts embedding mold
49
Consist of a series of interlocking plates resting on a
flat metal base, forming several compartments.
● Has the advantage of embedding more specimens at a time.
● Dividers are placed in squares to fit the tissue making
it a tissue block.
compound embedding unit
50
Used in positioning histological tissues accurately in
base molds.
● Compatible with most commonly-used processing and storage systems.
● Rings are precision-molded from premium-grade,
chemically-inert, high impact polystyrene for
dimensional rigidity and sturdiness.
plastic embedding ring and base mold
51
Embedding rings are
not reusable, and it
comes with the tissue
block
EMBEDDING RINGS
52
Replacement of
embedding ring:
cassette
53
Molds are placed
inside the embedding
ring and the tissue
goes down directly to
the mold. Then, it will
be filled up by
paraffin.
embedding ring
54
reusable.
● The lid is disposable.
○ Stays with the
tissue, can’t be
disposed without
disposing tissue.
BASE MOLDS
55
Made from thick paper or cardboard paper.
○ Laminated paper like from the magazines.
● Cheap to make and allow blocks to be stored without being removed.
● Provide easy and accurate identification of
specimens, thereby avoiding confusion and
interchange of tissue blocks.
● Not reusable.
paper boat
56
Convenient molds for busy routine laboratory, one
block being embedded in each compartment.
● Blocks are easily removed by flexing the plastic trays and by smearing the inside of the mold with glycerin or liquid paraffin.
plastic ice tray
57
Once the wax has solidified, the plastic walls
are peeled off one at a time, giving perfect
blocks that require no trimming.
○ They can be placed directly in the chuck of
the microtome.
peel a way
58
Ideal for embedding fragmentary biopsies.
● Not essential to smear them with glycerin.
● However, blocks are hard to remove.
watch glass
59
Used for small fragments which have been processed
(e.g. Bone marrow) which concentrates them without
the damage caused by orientation with forceps.
test tubes
60
● Disadvantage: often necessary to break the tube to remove the block.
test tubes
61
Used for embedding tissue intended for EM
microscopy.
epon resin
methacrylate plastic resin
62
All layers in
transverse sections
tubular tissues
63
All layers should
come
skin
64
Keep in center
endometrial cutting
65
Keep diagonally
long tissues
66
All layers should
come
intestine
67
All layers should
come
intestine
68
Swiss
membrane
69
Low Viscosity Nitrocellulose is highly explosive.
true
70
Blocks are easily removed by smearing the inside
of the mold with glycerin.
true
71
Tissues should not reach the edges during
embedding.
true
72
The removal of excess wax from the paraffin block to
expose the tissue surface in preparation for actual
cutting.
trimming
73
Sometimes referred to as ‘facing’ or ‘rough cutting’
trimming
74
End product of sectioning:
tissue ribbon
75
Process of placing the tissue ribbon to the
slide
fishig out in the water bath
76
Floatation water bath is 3-5 degrees lower
than the melting point because it allows the
paraffin together with the tissues to
smoothen, causing the disappearance of the
wrinkles,
TRUE
3 to 5 degrees
77
The most desirable block face does not exceed
0.5mm.
TRUE
78
The sides, top and bottom of the tissue block are
trimmed until perfectly level and all sides are parallel,
almost to the edge of the tissue.
TRUE
79
The paraffin between the sample and surface of the
block needs to be removed by trimming, first by hand
and latter with the microtome by making sections until
the first section with tissue appears.
TRUE
80
An _______ may be used for this procedure,
but it must still be relatively sharp to avoid damage to
the tissue.
old knife or blade
81
We must "sculpt" the paraffin block in order to make a
truncated pyramid with a trapezoidal upper surface,
the surface to be cut.
TRUE
truncated pyramid with a trapezoidal upper surface,
82
Tissue block has a definite shape:
truncated
83
○ Truncation is needed for ease in sectioning.
TRUE
84
It should be trapezoidal because the longest edge of
the upper surface is the first to be cut, whereas the
smaller edge will be the last.
TRUE
TRAPEZOIDAL
85
Both edges need to be parallel. In this way, the large
edge for a new section displaces the previous section,
which is attached to the blade by the shorter edge.
TRUE
PARALLEL
86
Paraffin blocks are cooled prior to trimming.
TRUE
87
Done on the microtome at approximately 15-30
microns at a time until the entire tissue surface is
exposed.
COARSE TRIMMING
15-30 microns
88
Depending upon the size and orientation of the tissue
● sample, shave conservatively into the block surface
taking appropriate cuts that may measure between
4-60 mm.
● Samples of small biopsy tissue may be trimmed only
to the depth of the first representation of several
levels that will be collected.
● When using the coarse feed, avoid cutting
unintentional thick sections as this will damage the
knife and possibly the block face.
COARSE TRIMMING
89
agressive trimming will cause
moth hole artifacts
90
moth hole artifacts are caused by
aggresive trimming
91
After coarse trimming, a _______ is held
between the tissue block and the block holder until the
wax begins to melt. The spatula is then withdrawn and
the block is gently pressed into position.
TRUE
heated spatula
until wax begins to melt
92
The block is allowed to harden for cutting proper by
facing them down in ice cold water or refrigerator for
________
5-10 minutes.
93
Placing blocks in a freezer can cause ________, where the friable tissue
separates from the surrounding wax
cohesive sections become difficult to obtain.
surface cracking
94
Re-chilling of the block may be required if the block
face becomes warm or if deeper levels are required.
TRUE
95
May be done by either setting the thickness adjuster
at 15 mm or by advancing the block using the coarse
feed mechanism.
FINE TRIMMING
96
The knife is usually tilted at 0-15° angulation on a
microtome to allow a clearance angle between the
cutting facet and the tissue block.
FINE TRIMMING
97
require smaller clearance angles
than wedge-shaped knives.
BICONCAVE KNIVES
98
Clearance angle and the tissue section:
indirect relationship
99
The higher the clearance angle, the thinner the
section.
TRUE
HIGHER, THINNER
100
The lower the clearance angle, the thicker the
section.
TRUE
lower, thicker
101
Bevel angle and the blade:
indirect
102
The lesser the bevel angle, the sharper the
blade will be.
lesser, shraper
103
Problem:
On trimming, tissue
smells of clearing
agent.
Reason: Clearing agent not
completely
removed due to
insufficient
Impregnation.
104
Problem: Tissue shrinks
away from wax
when trimmed.
Insufficient dehydration
Repeat the whole
procedure.
105
Problem:
On trimming, wax
appears
crystalline.
Contaminated wax
Block not cooled
rapidly enough
106
Problem:
On trimming, wax
appears
crystalline.
Contaminated wax
Block not cooled
rapidly enough
107
Excess plastic surrounding the tissue must be trimmed
away in a fashion that will yield a square or
rectangular sections.
TRUE
SQUARE OR RECTANGULAR
108
The capsule mold must be trimmed to a pyramid
where the pyramid tip and sides are exposed tissue.
TRUE
PYRAMID -capsule
109
The angle of the pyramid sides (facets) should be
about
45
110
In general, the smaller the block, the easier it will be
to section.
TRUE
SMALLER, EASIER
111
The most desirable block face does not exceed
0.5 mm
112
The ideal shape of of trimmed blocks
trapezoid
113
The top and bottom edges of the block must be
parallel
TRUE
PARALLEL
114
The left and right sides of the block are angled so that
the top edge is shorter than the bottom edge.
TRUE
TOP EDGE SHORTER THAN BOTOTM
115
is the process of cutting the
tissue block into slices or sections.
sectioning
116
Sections are usually cut from ______um in thickness for
routine histologic procedures with the use of a
microtome.
4-6 thickness um
117
most common type of microtome
ROTARY
118
set at a
temperature of 5°C-10°C lower than the melting point
of paraffin wax
floatation water bath
119
If the sections are too wrinkled, the ______
alcohol solution to the water bath can remedy this
prior to placing it onto the slide.
addition of 70%
120
Smoothened tissue will be properly oriented on the
slide by submerging the slide into the water bath at
approximately ________and directly under the
section.
45° angle
121
usually, is added in the water bath. However, this
technique is avoided as it enhances the growth of
bacteria and fungi in the floatation water bath.
GELATIN
122
may be used by simply dipping
the slide into the albumin solution or by subbing (with
the use of ulnar and smallest finger).
albumin
123
The slide with section should be drained in a _______
position on a paper towel prior to placing them onto a
warming plate or slide dryer to prevent the production
of air bubbles under the tissue and thus prevent
decreasing the adhesion of section to the slide.
VERTICAL
124
Warming plate or slide dryer should have a
temperature of 37°C-40°C (warm overnight) or 58°C
(20-30 minutes warming
125
Warming plate or slide dryer should have a
temperature of 37°C-40°C (warm overnight) or 58°C
(20-30 minutes warming
126
Discard the incomplete section and pick up one end of
the complete section either with a camel's hairbrush, a
pair of forceps, or the fingers.
TRUE
CAMELS HAIRBRUSH
FORCEPS
FINGERS
127
Float out a section on a floatation water bath set at
45°C-50°C(10°C lower than the melting point of wax).
128
Place the glass slide with the tissue section in an oven
to dry. The oven should be
37
129
process of coloring the transparent
tissue sections for the purpose of optically
differentiating the cells and tissue constituents of the
specimen microscopically.
STAINING
130
reversal process
used what
deparaffinization with clearing agents
131
use of descending grades of alcohol
rehydration
132
The most common staining method used in the
histopathology laboratory is the
This method is used as a routine
staining technique as it can easily color the cytoplasm,
nucleus, and organelles.
Hematoxylin and
Eosin(H&E) method.
133
collagenous connective tissue of the skin;
MASSONS
134
stain Pneumocystis or Aspergillus spp. in the
lungs;
GMS
135
stain glycogen,
glycoproteins, and proteoglycans
PAS
136
to stain muscle fibers,
collagen, and nuclei, among others
COMORI
137
Dehydrate with ascending grades of alcohol,
TRUE
ASCENDING
