histopath Flashcards
(257 cards)
1
Q
Squash
preparation /
Crushing
A
Process where small pieces of tissue not more than
1mm in diameter are placed in a microscopic slide and
forcibly compressed with another slide or with
coverglass .
Vital dyes are placed at the slide and coverslip junction
and absorbed through capillary action .
2
Q
Frozen section
A
- Normally utilized when a rapid diagnosis of the tissue
in question is required , and especially recommended
when lipids and nervous tissue elements are to be
demonstrated .
3
Q
Smearing
A
Useful in cytological examinations , particularly for
cancer diagnosis .
4
Q
Fixation
A
Preservation
5
Q
Decalcification
A
Calcium or lime salts are removed from the tissues
Optional process : for calcified tissues only such as
bones and teeth
6
Q
Dehydration
A
- Desiccation
Removing intracellular and extracellular fluid / water
7
Q
Clearing
A
De - alcoholization
Removing alcohol used in dehydration
8
Q
Infiltration
A
Impregnation
9
Q
Embedding
A
Casting or Blocking
10
Q
Trimming
A
Removing excess wax from the tissue block
Optional process : not all tissue blocks have excess wax
11
Q
Sectioning
A
Section cutting
- Cutting tissue blocks into uniformly thin slices
12
Q
Staining
A
Dyeing
13
Q
Labelling
A
- Proper labelling
14
Q
Smearing
Technique
A
Material
15
Q
a . Streaking
A
Applicator stick
or platinum
loop .
16
Q
b . Spreading
technique
A
Applicator stick
to tease the
mucous strands
to make a
moderately
thick film .
17
Q
c . Pull - apart
technique
A
Slides facing
each other as a
drop of
secretion is
sandwiched in
between .
18
Q
d . Touch
preparation
or
Impression
smear
A
One slide .
19
Q
Autolysis
A
the destruction of the tissues ( breaking down of the
protein of the cell ) by enzymes which are produced by
the tissues and eventually liquefy it .
It is the first to occur among all post - mortem changes
20
Q
Putrefaction or
Decomposition
A
the decomposition of organic matter under the influence
of microorganisms accompanied by the development of
disagreeable odors .
21
Q
Degeneration
A
- a retrogressive pathologic process in cells in which the
cytoplasm undergoes deterioration while the nucleus is
preserved .
22
Q
Two Basic
A
Mechanisms in Fixation
23
Q
- Additive
A
The chemical constituent of the
fixative is take in and becomes
part of the tissue through cross - link
formation or molecular complexes .
24
Q
- Non
additive
A
The fixing agent is not incorporated
into the tissue but alters the tissue
composition and stabilizes it
through water removal .
New cross - links are formed
preventing autolysis and bacterial
decomposition .
25
Main Factors Involved
in Fixation
26
1. Hydrogen lon
Concentration
pH : 6.0-8.0
Average : 7.0 ( neutral pH )
27
2. Temperature
-
Traditional / usual : Room temperature ( 18-30 ° C )
Tissue processors : Autotechnicon ( 40-42 ° C )
Electron Microscopy and Histochemistry : 0-4 ° C
O Mast cells for EM : Room temperature
Nucleic acids fixation : Rapid at higher temperature
Formalin heated to 60 ° C → rapid fixation of very
urgent biopsy specimens
Formalin heated to 100 ° C → to fix tissues with
tuberculosis
28
3. Thickness of
sections
Small
O
Electron Microscopy : 1-2 mm²
Light Microscopy : 2 cm²
Thin
O Light Microscopy : ≤0.4 cm or as prescribed by
tissue processor manufacturer
Large solid tissue , such as uterus , should be
opened or sliced thinly
O Brain is usually suspended whole in 10 % Neutral
Buffered Formalin for 2-3 weeks .
29
4. Osmolality
-
Best results are obtained using slightly hypertonic
solutions ( 400-450 mOsm )
Hypertonic solutions - Shrinkage =
Isotonic ( 340 mOsm ) / Hypotonic solutions
swelling and poor fixation
Added to Osmium tetroxide fixatives for EM : Sucrose
=
30
5. Concentration
-
-
Formaldehyde : 10 %
Glutaraldehyde : 3 %
Glutaraldehyde for Immunoelectron microscopy :
0.25 %
31
6. Duration
fixation
of
-
-
-
Most formalin fixatives : 24 hours ( washed out )
Buffered formalin : 2-6 hours up to 1 week
EM : 3 hours ( New books : 0-4 hours ; Average : 2 hours )
Prolonged fixation may cause shrinkage and
hardening of tissue
32
ROUTINE FORMALIN
FIXATIVES
33
1. 10 % Formol - Saline
Fixation time :
24 hours at 35 ° C
48 hours at 20-25 ° C
Traditionally , it is the most commonly used
fixative in pathology
Best fixative for central nervous tissues and
general post - mortem tissues for
histochemical examination .
34
2 . Calcium
formalin
Fixative )
acetate
( Lillies
- It is used to preserve phospholipids
Replaced formol - saline as the most
commonly used fixative in pathology
because :
Simple to prepare
Buffered to pH 7 by acetate
35
3 . 10 %
Buffered
Fixation time :
4-24 hours
Formalin / Phosphate
buffered Formalin
( pH 7 )
Neutral
-
Best general tissue fixative
Best Fixative for frozen sections
Recommended for surgical , post mortem
and research specimens .
Prevents precipitation of acid formalin
pigments .
Best fixative for iron - containing pigments
and elastic fibers .
36
4 . Formol corrosive /
Fixation time :
3-24 hours
Formol - sublimate /
Formol - Mercuric
Chloride
-
Recommended for routine post - mortem
tissues .
It is excellent for silver reticulum methods .
It fixes lipids , especially neutral fats and
phospholipids .
Forms black deposits .
No frozen sections are made
37
Alcoholic
Gendre's fixative
Formalin /
- It contains ethyl alcohol saturated with picric
acid .
It enhances immunoperoxidase studies for
EM if post - fixed with phenol formalin for 6
hours or more .
It fixes and dehydrates at the same time and
fixes sputum since it coagulates mucus
38
SPECIAL FORMALIN
FIXATIVES :
- Cajol's formol ammonium bromide - good
fixative for nervous tissue ( astrocytes ) .
Fixatives for acid mucopolysaccharides .
Baker's formol calcium used for the
preservation of lipids since most formalin
fixatives are inert to lipids .
-
39
1. Chromic acid
Used in 1-2 % , used as a constituent of a
compound fixative .
It precipitates all proteins and adequately
preserves carbohdyrates .
Formaldehyde must be added to chrome
containing tissues before use to prevent
counteracting effects and consequent
decomposition of solution upon prolonged
standing .
40
2 . Regaud's
Fluid /
Moeller's fluid
Recommended for demonstration of Chromatin ,
Mitochondria , Mitotic figures , Golgi bodies ,
RBC's and colloid - containing tissues
41
3 . Orth's Fluid
Fixation time :
36-72 hours
Recommended for study of early degenerative
processes and tissue necrosis
Demonstrates Rickettsia and other bacteria
42
4 . Potassium
dichromate
Preserves mitochondria ( pH 4.5-5.2 )
Fixes lipids
Used in 3 % aqueous solution
It fixes but does not precipitate cytoplasmic
structures .
43
Mercuric Chloride
Fixatives
44
1. Zenker's fluid
Fixation time :
12-24 hours
Mercuric chloride + glacial acetic acid just before
its use .
To prevent turbidity and formation of dark
precipitates
Good general fixative for all kinds of tissue
Recommended for fixing small pieces of liver ,
spleen , connective tissue fibers and nuclei
May act as mordant to make certain special
staining possible
Contains glacial acetic acid which makes the
solution unstable
45
2 . Zenker
formol / Helly's
Solution
Fixation time :
12-24 hours
" Bloody Helly "
Mercuric chloride + 40 % formaldehyde just
before its use .
It is an excellent microanatomic fixative for
pituitary gland , bone marrow and blood
containing organs such as the liver and spleen .
Better nuclear fixations and staining than Zenker's
It presevres cytoplasmic granules better than
Zenker's
Disadvantage : Brown pigments are produced if
tissues are allowed to stay in the fixative for more
than 24 hours due to RBC lysis .
Remedy : immerse the tissue in alcoholic picric
acid or sodium hydroxide
46
3 . Heidenhain's
Susa solution
Fixation time :
3-12 hours
- Recommended for tumor biopsies especially of the
skin
It is an excellent Cytologic fixative
Produces minimum shrinkage and hardening of
tissues due to the counter - balance of the swelling
effects of acid ( trichloroacetic acid ) and the
shrinkage effect of a metal ( mercury ) .
47
4 . Schaudinn's
Solution /
Sublimated
alcohol
A solution of mercuric
chloride , alcohol , and glacial acetic acid
Used on wet smears for cytologic examinations .
chloride , sodium
48
5 .
Fixation time :
1½ to 2 hours
( Rapid fixation )
B - 5 Fixative
Composed of mercuric chloride and sodium
acetate .
Commonly used for bone marrow biopsies .
Just prior to use , add 1 mL of 40 % formaldehyde
to 10 mL of B5
49
A. Bouin's solution
Fixation time :
6-24 hours
-
-
-
Fixation of embryos and pituitary biopsies
It is an excellent fixative for preserving soft and
delicate structures ( endometrial curettings )
Yellow stain is useful in fragmentary biopsies
Preferred fixative for Masson's trichrome
staining for collagen , elastic or connective tissue
50
B. Brasil's Alcoholic
Picroformol Fixative
-
Best routine fixative for glycogen
It is better and less messy than Bouin's solution
51
1. Flemming's Solution
Fixation time :
24-48 hours
The most commonly used Chrome - Osmium
acetic acid , recommended for nuclear
preparation of such sections .
Excellent for nuclear structures such as
chromosomes
Permanently fixes fats / lipids
52
2. Flemming's Solution
without Acetic Acid
Fixation time :
24-48 hours
Recommended for cytoplasmic structures
such as mitochondria
The removal of acetic acid from the formula
serves to improve the cytoplasmic detail of the
cell .
53
1 . Methyl Alcohol
100 %
Methanol )
( CH3OH /
- Used in Wright's stain as a diluent
Excellent for fixing wet and dry smears , blood
smears and bone marrow tissues
54
2 . Isopropyl Alcohol
95 % ( Isopropanol )
- It is used for fixing touch preparations
( impression smears ) , although some are air
dried and not fixed , for certain procedures
such as Wright - Giemsa staining .
55
3 .
Fixation time :
18-24 hours
Ethyl Alcohol
( C2H5OH / Ethanol )
-
-
Is used in 70-100 % concentrations .
Lower concentrations ( 70-80 % ) will cause
RBC lysis and inadequate WBC preservation
It fixes blood , tissue films and smears
Used for histochemistry especially for enzyme
studies
Can be both used as a simple and compound
fixative .
56
4 .
Fixation time : 1-3 hrs .
Carnoy's Fluid
-
The most rapid tissue fixative
Recommended for fixing chromosome , lymph
glands and urgent biopsies
It is used to fix brain tissue for rabies
diagnosis .
It preserves Nissl granules and very suitable
for Curettings ( small tissue fragments ) .
57
5 . Alcoholic
Formalin /
Gendre's
- Better preserves glycogen
Capable of coagulating mucus and is used as
a fixative for sputum cytology
58
6 . Newcomer's Fluid
Fixation time :
12-18 hours @ 3 ° C
- For fixing mucopolysaccharides and nuclear
proteins
It is both a nuclear and histochemical fixative .
59
Special Factors Affecting
Fixation
60
1. Size and thickness
Larger and thicker tissues require more fixative
and fixation time
61
2. Presence
mucus
of
Prevents complete penetration of fixative ,
hence , tissues that contain mucus are fixed
slowly and poorly
Excess mucus may be washed away with NSS .
62
3. Presence of fats
Fatty tissues should be cut in thin sections and
should be fixed longer .
63
4. Presence of blood
Tissues containing large amount of blood ( e.g.
Blood vessels and spleen ) should be flushed
out with saline ( arterial cannulization ) before
fixing .
64
5. Cold temperature
Inactivates enzymes
65
6. Hot temperature
Denatures enzymes
66
1. Size and thickness
Smaller and thinner tissues require less fixative
and shorter fixation time
67
2. Agitation
- Fixation is accelerated when automatic or
mechanical tissue processing is used .
Autotechnicon
68
3. Moderate heat
Accelerates fixation but hastens autolytic
changes and enzyme destruction
69
I. Acid Decalcifying
Agents
70
1. Nitric acid
The most common and fastest decalcifying agent
used so far
71
Recommended
concentrations :
Very rapid decalcifying agent , producing minimal
distortion
72
5-10 %
Recommended for routine purposes
73
a . 10 % aqueous
Nitric Acid
Decalcification time :
12-24 hours
Recommended for urgent biopsies and for needle
and small biopsy specimen
Used for large or heavily mineralized cortical bone
specimen
74
b . Formol - Nitric
acid
Decalcification time :
1-3 days
For urgent biopsies with good nuclear staining .
Yellow color imparted by nitrous acid is removed
through neutralization with 5 % sodium sulfate and
running tap water for 12 hours
75
C. Perenyi's fluid
Decalcification time :
2-7 days
-
It is recommended for routine purposes .
It decalcifies and softens tissues at the same time
Maceration is avoided due to the presence of
chromic acid and alcohol
76
d . Phloroglucin
Nitric Acid
Decalcification time :
12-24 hours
Most rapid decalcifying agent , recommended for
urgent works
Has poor nuclear staining
Yellow color formation
77
2. Hydrochloric
acid / Muriatic
acid
Inferior compared to nitric acid ( slower action and
greater distortion of tissue )
Good nuclear staining
Recommended for surface decalcification of
tissue blocks if used in 1 % solution with 70 %
alcohol
Von Ebner's fluid
Formula : 36 % Sat. Aq . NaCl , Conc . HCI , Distilled water
78
1. Failure to arrest early cellular
autolysis
Due to the failure to fix immediately by
which one first allowed the tissue to dry
before fixing or insufficient fixative
79
2. Too brittle and too hard blocks
Due to prolonged fixation
80
3. Soft and feather - like tissues
Due to incomplete fixation
81
4. Removal of fixative soluble
substances
Wrong choice of fixative
82
5. Presence of artefact pigments on
sections
Incomplete washing of fixative
83
6. Shrinkage and swelling of cells in
tissue blocks
Due to overfixation
84
7. Enzyme inactivation and loss
Wrong choice of fixative
85
Incomplete fixation → Improper and
incomplete clearing and impregnation →
86
3. Formic acid
Moderate - acting decalcifying agent
Recommended for routine decalcification of post
mortem research tissues
87
Decalcification time :
2-7 days
Disadvantage : Not suitable for urgent examination
88
4. Trichloroacetic
acid
-
Does not require washing out
Not recommended for urgent examinations ( very
slow acting )
89
5. Sulfurous acid
- Weakest decalcifying agent , suitable only for
minute pieces of bone
90
6. Chromic acid
Used as a fixative and decalcifying agent
Caution : It is an environmental toxin , highly
corrosive to skin and mucous membrane and
carcinogenic
91
7. Citric acid
Citrate buffer
Does not produce cell or tissue distortion
92
3 Ways to Measure
the Extent of Decalcification
93
1. Physical or
Mechanical
Method
By touching or bending the tissue with the fingers to
determine the consistency of tissues
By pricking the tissue with a fine needle / probe
94
2. X - ray /
Radiological
method
- Very expensive , although it is the most ideal , most
sensitive and most reliable method
Good but not always convenient
Not recommended for mercuric chloride - fixed tissues
( radio - opacity will interfere with the plate interpretation )
95
3. Chemical
method /
Calcium
Oxalate test
-
1. Concentrated Ammonium Hydroxide
2. Saturated Aqueous Ammonium Oxalate
Detection of calcium in acid solutions by precipitation
of insoluble calcium hydroxide or calcium oxalate
Presence of cloudiness indicates the presence of Ca
( incomplete decalcification )
Simple , reliable and convenient method recommended
for routine purposes ( still favoured )
Decalcifying fluid is changed every 24-48 hrs
Solutions used :
96
a . Ethanol / Ethyl
alcohol
- Undoubtedly the best dehydrating agent ( fast acting ,
mixes with water and organic solvents and penetrates
tissues easily )
Has the advantage of not being poisonous and not
very expensive
Should be at least 99.7 % pure
97
b . Isopropanol /
Isopropyl
alcohol
Should be used if good - grade absolute ethyl alcohol is
not easily available
98
c . Methanol /
Methyl alcohol
Toxic dehydrating agent
For blood and tissue films and for smear preparations
99
d . Butanol / Butyl
alcohol
Utilized for plant and animal microtechniques .
100
5. Cedarwood oil
Clearing time :
2-3 days
-
Clears both Paraffin and Celloidin sections
Recommended for CNS tissues and cytological
studies ( esp . Smooth muscles and skin )
Very expensive and it requires 2 changes in
clearing solution
Quality is not always uniform and good and is
extremely slow
It becomes milky on prolonged storage
101
6. Aniline oil
Recommended for clearing embryos , insects
and very delicate specimens since it clears
70 % alcohol without excessive tissue shrinkage
and hardening
102
7. Clove oil
-
It removes aniline dyes and dissolves Celloidin ;
Tissues become brittle
Its quality is not guaranteed due to its tendency
to be adulterated
Not suitable for routine purposes because it is
expensive
103
8. CC14 ( Carbon
Tetrachloride )
-
Properties are very similar to chloroform but it is
cheaper
Toxic on prolonged exposure
104
9. Tetrahydrofuran
- Dehydrates and clears at the same time since it
is miscible in both water and paraffin
105
10. Methyl
benzoate /
Methyl
Salicylate
These are slow - acting clearing agents that can
be used when double embedding techniques
are required .
106
2. Acetone
( CH3COCH3 )
A cheap , rapid dehydrating agent utilized for
most urgent biopsies ( 1/2 to 2 hours )
Its use is limited only to small pieces of tissues
due to its extreme volatility and flammability
107
3. Dioxane /
Diethylene
Dioxide
Excellent dehydrating and clearing agent
miscible to water , melted paraffin , alcohol and
xylol / xylene
Expensive and toxic and extremely dangerous
( main disadvantage )
108
4. Cellosolve /
Ethylene glycol
monoethyl ether
Dehydrates rapidly and not harmful to tissues
Toxic by inhalation , skin contact and ingestion
( use propylene - based glycol esters )
109
5. Triethyl
phosphate
Tissues can be transferred directly after fixation
& washing
Used to dehydrate sections and smears
following certain stains
110
6. Tetrahydofuran
Dehydrates and clears tissues since it is
miscible to water and paraffin
It is toxic if ingested or inhaled . Vapors cause
nausea , dizziness and headache .
It is an eye and skin irritant and prolonged
exposure ( up to 6 months ) may cause
conjunctival irritation .
111
Commonly Used
Clearing Agents
112
Clearing time :
½ to 2 hours ( usual )
15-30 Minutes
( urgent biopsies )
1. Xylene
Advantages :
An excellent and true clearing agent
1 .
2 .
3 .
4. Can be used with celloidin sections
Disadvantages :
1. It is highly flammable
The most rapid clearing agent
Cheap and does not extract out aniline dyes
It is miscible with absolute alcohol and paraffin
2. When dehydration is not complete , the xylene
becomes milky when the tissue or section is
added to it
113
2. Toluene
Clearing time :
1-2 hours
Substitute to xylene or benzene
It is miscible with absolute alcohol and paraffin
It is not carcinogenic but highly concentrated
emit fumes that are toxic upon prolonged
exposure
It acts slower than benzene and is expensive
114
3. Benzene
Clearing time :
15-60 minutes
-
Rapid acting , recommended for urgent biopsies
and routine purposes
It is miscible with absolute alcohol and paraffin
It is highly flammable
Carcinogenic or may damage bone marrow
( Aplastic anemia ; If antibiotic : Chloramphenicol )
115
4. Chloroform
Clearing time :
6-24 hours
-
It is the best of the traditional clearing agents for
routine use
Gives the widest latitude
It is recommended for tough ( skin , fibroid and
decalcified tissues ) and large tissue specimens
Also best for nervous tissue , lymph nodes ,
granulation tissue , and fetal and other delicate ,
highly cellular specimens
116
3 Ways of Paraffin
Wax Impregnation ( and Embedding )
117
1. Manual
processing
Requires at least 4 changes of wax with 15 minutes '
interval
Total : 1 hour
118
2. Automatic
Processing
Makes use of an automatic tissue processing machine
( e.g. Autotechnicon ) which fixes , dehydrates , clears
and infiltrates tissues , thereby decreasing the time
and labor needed during the processing of tissues ,
resulting in a more rapid diagnosis with less
technicality
Autotechnicon / Elliot - Bench Type Tissue Processor :
Fixation , Dehydration , Clearing , Infiltration
119
3. Vacuum
Embedding
Impregnation under negative atmospheric pressure
Recommended for urgent biopsies and delicate
tissues such as lung , brain , connective tissues ,
decalcified bones , eyes spleen and CNS .
Reduces 25-75 % of impregnation time
Most rapid
120
Several Types
of Blocking - out Molds
121
1. Leukhart's
embedding
mold
Consist of 2 L - shaped pieces of heavy brass or metal , a
base being formed by a piece of 1/8 inch thick copper
brass , about 3X2 inches square , or a piece of plate glass .
122
2. Compound
embedding
mold
Made up of a series of interlocking plates resting on a flat
metal base , forming several compartments .
123
3. Plastic
embedding
rings with
base mold
Consist of a special stainless steel base mold fitted with a
plastic embedding ring which later serves as the block
holder during cutting .
Example : Tissue Tek
124
4. Disposable
Embedding
Molds
a . Peel - aways
o available in 3 different sizes , peeled off one at a time
b . Plastic Ice Trays
o Used in ordinary refrigerators recommended for busy
routine laboratories
c . Paper boat
o Cheap and easy to make
o Used for embedding celloidin and paraffin blocks
125
a . Wet Celloidin Method
Recommended for bones , teeth , large
brain sections and whole organs
Tissues must be cut wet ( both the knife &
tissue block are kept moist with 70 %
alcohol while cutting )
126
b . Dry Celloidin Method
* Gilson's mixture -equal
parts of chloroform and
cedarwood oil
- Preferred for processing of whole eye
sections
Material embedded with the dry method
can be cut without alcohol due to the
presence of the cedarwood oil in the block
127
Reagent to Tissue Ratio
Step
128
20 : 1
Fixation
129
10 : 1
Dehydration
130
25 : 1
Infiltration
131
1. SLIDING
MICROTOME
1789 : Adams
a . Base - Sledge Microtome
b . Standard Sliding Microtome
For cutting celloidin embedded sections
Favored in laboratories where very hard tissue
or blocks are sectioned
Most dangerous because of its movable
exposed knife
The block remains stationary while the knife is
moved backward and forward
132
2 . ROTARY
MICROTOME
1885-1886 : Minot
For cutting paraffin embedded sections
Most popular and most common type used for
routine and research studies
133
3. ROCKING
MICROTOME
AKA : Cambridge
1881 : Paldwell &
Trefall
The simplest
For cutting serial sections of large blocks of
paraffin embedded tissues
134
4 . FREEZING
MICROTOME
1848 : Queckett
For cutting unembedded frozen sections
Replaced in most laboratories by the cryostat ,
which is easier to operate ( faster and gives
thinner sections ) .
135
5. ULTRATHIN
MICROTOME
For cutting specimens into extremely thin
slices ( 0.5u ) for electron microscopy work
136
1. Carborundum hone
Available in a wide variety of fineness
Used only for badly nicked knives
137
2. Arkansas stone
A stone of medium fineness
138
3. Yellow Belgian / Belgium
- The finest ( best result )
139
4. Belgian Black Vein
-
Oils used :
a .
b .
Also called oil stones , because oil is
commonly used as a lubricant
C.
d .
e .
light machine oil
Liquid paraffin oil ( mineral oil )
Vegetable oil
Xylene - tends to evaporate too rapidly
Natural oil - become messy
140
1. PLANE CONCAVE
( 25mm in length )
Frok A
Planoconcave
One side of the knife is flat / straight ( Celloidin
tissues ) while the other is concave ( Paraffin
tissues )
Base - sledge / Rocking / Rotary microtome
141
2. BICONCAVE
( 120mm in length )
Profile B
Biconcave
With both sides concave ( Paraffin sections )
Rotary microtome
142
3. PLANE WEDGE
( 100 mm in length )
Paris . C
Wilge shaped
Have both sides straight ( frozen sections and
extremely hard and tough specimens )
Base - sledge / Sliding microtome
143
1. PARAFFIN SECTION
( 4-6 μ thick )
- For paraffin embedded tissue blocks
May be cut by rotary and rocking microtome
144
2. CELLOIDIN SECTION
( 10-15μ thick )
-
For celloidin embedded tissues
Usually used by means of the sliding
microtome
145
3. FROZEN SECTION
May be cut from tissues that have been
fixed and frozen with CO2 ( cold knife
procedure ) or for fresh or fixed tissues
frozen with the cryostat ( cold microtome )
146
Special Processing
Techniques
147
1. Freeze
Drying
-
Preserving tissues by rapid freezing ( Quenching ) and
removing water ( Dessication ) by a physical process
from the still frozen tissue block without the use of any
chemical fixative .
Tissue size : 2 mm thick
Complete processing time : 24-48 hours
Advantages :
1. Produces minimum shrinkage
2. Allows tissues to be processed in a fresh state
3. Less displacement
148
2 . Freeze
Substitution
-
Similar to freeze - drying
Difference : Tissue is fixed in Rossman's fluid or in
1 % acetone and dehydrated in absolute alcohol
Rossman's fluid : Satrurated Picric acid and
Formaldehyde
149
a . Hematoxylin
The most important and the most commonly used for
routine histologic studies
150
b . Orcein
A vegetable dye extracted from lichens which are
normally colorless , but when treated with ammonia and
expose to air , produce blue or violet colors
Mainly used for staining Elastic fibers
151
c . Cochineal
An old histologic dye extracted from the female Cochineal
Bug ( Coccus cacti )
152
d . Saffron
A plant with orange stigmas yielding a dye
153
13. Crystal Violet
- A nuclear or chromatin stain used for staining
amyloid in frozen sections and platelets
Gentian Violet Mixture of crystal violet , methyl
violet and Dextrin
154
14. Giemsa
Used for staining blood to differentiate
leukocytes
155
15. Gold Sublimate
Used for metallic impregnation , made up of gold
chloride and mercuric chloride
156
16. lodine
a . Gram's
b . Lugol's
- The oldest of all stains
Stains amyloid , cellulose , starch , carotenes and
glycogen
157
17. Janus Green B
Used for demonstrating mitochondria ( intravital
staining )
158
18. Malachite Green
Used as a contrast stain for staining ascaris
eggs and erythrocytes
Used as bacterial spore stain
159
19. Methyl Green
- Stains chromatin green in the presence of an
acid
160
20. Methylene Blue
Plasma cells , Fresh sputum for malignant cells ,
evaluation and differentiation of bacterial
organisms , Diphtheria diagnosis and Nervous
tissue vital staining
161
21. Loeffler's
Polychrome
Methylene Blue
- Used in frozen sections for rapid diagnosis
162
22. Methylene Violet
- Coloring nuclei of leukocytes reddish - purple in
the presence of methylene blue
163
23. Neutral Red
- For observing cell granules and vacuoles of
phagocytic cell
164
24. Night Blue
Substitute for carbol fuchsin in acid - fast staining
165
25. Orcein
- An excellent stain for Elastic fibers ( Taenzer
Unna Orcein Method ) ; Demonstrates the finest
and most delicate fibers in skin ( Dermatological
studies )
166
26. Osmium
Tetroxide
- Used as a fixative ; Used to stain fats
167
27. Prussian Blue
Normally utilized for the manufacture of paints ;
Used as microanatomical contrast stain ; For
demonstration for the circulatory system by
injection ( intravital staining )
168
28. Rhodamine B
Used with osmic acid to fix and stain blood and
glandular tissues
169
29. Silver Nitrate
- Used in identification of Spirochetes , reticulum
and other fiber stains
170
30. Toluidine Blue
- Recommended for staining of Nissl granules or
chromophilic bodies ; Nuclear stain for fixed
tissues ; Used as a substitute for thionine in fresh
tissue sections
171
31. Victoria blue
- For demonstration of neuroglia in frozen
sections
172
1. Cytoplasm & proteins in edema fluid
Pale pink
173
2 . Decalcified bone
Osteoid
Collagen
Pink
174
3 . Muscle fiber
Deep pink
175
4 . RBC
Eosinophil granules
Keratin
Bright orange to Red
176
5 . Calcium & Calcified bone
Purplish blue
177
6 . Plasma cells
Osteoblast
Basophilic cytoplasm
Purplish pink
178
7 . Karyosome
Dark blue
179
8 . Nucleus
Blue to Blue - Black
180
9 . Cartilage
Light blue to Dark Blue
181
1. Van Gieson's
( Acid fuchsin picric
acid )
Mixture of picric acid and acid fuchsin for the
demonstration of Connective tissues , mucin
and Elastic tissue
182
2. Acridine Orange
A basic acridine fluorochrome which permits
discrimination between dead and living cells ,
giving GREEN fluorescence for DNA and a RED
fluorescence for RNA
183
3. Acridine Red 3B
For calcium salts and phosphatase activity
184
4. Alcian Blue
- Stain acid mucopolysaccharides ; More specific
for connective tissue and epithelial mucin
185
5. Aniline blue
- A cytoplasmic stain used for counterstaining of
Epithelial sections
186
6. Basic Fuchsin
a . Carbol fuchsin
b . Coleman's feulgen
c . Schiff's
d . Mallory's fuchsin
e . Aldehyde fuchsin
( Gomori's )
- A plasma stain utilized also for deep staining of
acid fast organisms , mitochondria and
differentiation of smooth muscles with the use of
picric acid
187
7. Benzidine
Used for staining haemoglobin
188
8. Bismarck brown
Use for staining diphtheria ; Used as a contrast
stain for Gram's technique , acid fast and
Papanicolau method .
189
9. Carmine
-
Used as a chromatin stain for fresh materials in
smear preparations
Best Carmine Combined with aluminium
chloride to stain glycogen
=
190
10. Mayer's
Carmalum solution
- A mordanted dye acting as a basic dye and
staining acid substrances
191
11. Celestine Blue
Recommended for routine staining of fixed
sections
192
12. Congo Red
Best known as indicator ; May be utilized as a
stain for axis cylinders in embryos ; Used for
staining elastic tissues , amyloid and myelin
( Krajian's method )
193
RED
Yellow
194
Eosin Y
Eosin B
Phloxine B
Rose Bengal
Picric acid
Orange G
195
Red
Blue
196
Neutral red
Methylene blue
197
Safranin
Toluidine Blue
198
Carmine
Celestine Blue
199
LYSOCHROMES
( Oil Soluble Dyes )
200
1. Sudan Black
B ( SBB )
Greatest affinity for phospholipids or neutral fats
( triglycerides )
A more sensitive coloring agent than other
lysochromes
Should be discarded if the brownish black color
appeared .
Demonstrates lipids that are resistant to paraffin
embedding
201
2. Sudan IV or
Sharlach R
Recommended for neutral fats ( triglycerides )
Do not color phospholipids and fine lipid droplets
202
3. Sudan III
-
First Sudan dye introduced into Histochemistry
Fat soluble
A good stain for the CNS
203
a . Water
Low refractive index ; good only for temporary
mounting
-
204
b . Glycerin ( RI : 1.47 )
Also used as preservative , has high index of
refraction
-
205
c . Farrant's Medium
( Gum syrup ) ( RI : 1.43 )
Does not solidify upon storage -
206
d . Apathy's Medium
Used for Methylene Blue - stained nerve
preparations and as general purpose aqueous
mountant
207
e . Brun's Fluid
- Recommended for mounting frozen sections from
water or paraffin sections which require
dehydration and clearing
208
a . Canada Balsam
( RI : 1.524 )
- Natural resin extracted from the Canadian Tree ,
Abus balsamea
- Recommended for whole mounts and for thick
sections because it does not shrink much
Miscible with xylene and is quite expensive
209
b . DPX ( RI : 1.532 )
- Recommended for small tissue sections
210
c . XAM ( RI : 1.52 )
Synthetic resin mixture in xylene in pale yellow or
colorless solution
211
d . Clarite ( RI : 1.544 )
- Synthetic resin which is soluble in xylene ( usually
diluted to 60 % with xylene )
212
1. Mayer's Egg Albumin
Most commonly used because it is very
easy to make and relatively inexpensive
Formula : Egg white ( 50 mL )
Glycerin ( 50 mL )
Thymol crystals ( 100 mg )
213
2. Dried Albumin
Formula : Dried albumin ( 5 g )
Sodium chloride ( 5 g )
Distilled water ( 100 mL )
Thymol crystals
214
3. 1 % Gelatin
Formula : Gelatin ( 1 g )
Distilled water ( 100 mL )
Glycerol ( 15 mL )
Phenol crystals ( 2 g )
215
4. Gelatin
Formula : 1 % Gelatin ( 5 mL )
2 % Formaldehyde ( 5 mL )
216
5. Starch Paste
Formula : Powdered starch ( 1 g )
Distilled water ( 30 mL = 10 mL
cold , 20 mL boiling )
Hydrochloric acid ( 2 drops )
Thymol crystals
217
6. Plasma
Readily available from outdated blood
stored in blood banks , dispensed into
sterile tubes of 0.5 mL each .
218
7. Poly - L - Lysine
0.1 % aqueous detergent solution which
is further diluted 1:10 with distilled water
prior to use ( Final dilution : 0.01 % ) .
Used as a section adhesive in
immunohistochemistry .
219
8. 3 - APES ( 3
aminopropyltriethoxysilane )
- Very useful in cytology particularly for
cytospin preparations of proteinaceous
or bloody material .
220
1. Keratin
Highly sensitive marker for epithelial cells
( Epithelial tumors )
221
a . Cytokeratin 7
( CK7 )
Carcinomas of the lung , breast , uterus and
ovaries ( serous tumors )
222
b . Cytokeratin 20
( CK20 )
Carcinomas of colon and stomach
223
c . CK 7 and CK20
- Transitional carcinomas of the bladder and
mucinous ovarian tumor
224
2. Epithelial membrane
antigen ( EMA )
-
Determining site tumor ,
Breast , lung and kidney adenocarcinomas
225
3 . Carcinoembryonic
membrane antigen
( CEA )
Differentiating
mesothelioma
GIT , pancreas , lung , breast , ovary , uterus
and cervix
Thyroid , lung and neuroendocrine tumors
adenocarcinoma and
226
4. Thyroid
transcription factor
1 ( TTF - 1 )
- Distinguishing lung adenocarcinoma from
mesotheliomas
227
5. Prostate specific
antigen ( PSA )
- Prostatic
salivary gland tumor .
carcinoma ; pancreatic and
228
1 . Actin
Muscle differentiation ( smooth , skeletal and
cardiac muscle )
229
2 . ntin
Melanomas and Schwannomas
230
3 . Desmin
- Myogenic tumors
rhabdomyosarcoma )
( leiomyoma and
231
4 . Glial fibrillary acidic
protein ( GFAP )
- Astrocytoma
232
5. Neurofilament ( NF )
- Neuronal or neuroendocrine differentiation
233
6 . S100 protein
- Calcium - binding protein that is expressed in
CNS glial cells , Schwann cells ,
melanocytes , chondrocytes , skeletal and
cardiac muscle and myoepithelial cells
234
1. Neuron - specific
enolase ( NSE )
Neural or neuroendocrine differentiation
235
2. Chromogranin
Neuroendocrine differentiation
236
3. Synaptophysin
Associated with presynaptic vesicles of neurons ;
identified in normal neurons and neuroendocrine
cells .
237
1. Human chorionic gonadotropin
( HCG )
Choriocarcinoma
238
2. Alpha - fetoprotein ( AFP )
- Endodermal sinus tumors
( yolk sac differentiation ) ;
Hepatocellular carcinomas
239
3. Placenta - like alkaline phosphatase
( PLAP )
Germ tumors
( Germinomas )
cell
240
1. Muscle - specific actin and myosin ;
Myo - D1 , myoglobin and myogenin
Myogenic tumors ( Skeletal
muscle )
241
2 . CD68 or FAM 56 ; alpha - 1
antiptrypsin and alph - 1
antichymotrypsin
Fibrohistiolytic tumors
242
3 . Endothelial markers : Factor VII
related antigen , CD31 and Ulex
Europaeus ( UEA )
Vascular
( Angiosarcoma )
tumors
243
4 . $ 100 protein
Melanosome ( HMB - 45 )
Melan - A ( MART - 1 )
Melanomas
244
5. Best : Leukocyte Common Antigen
( LCA ) or CD45
Lymphomas
245
SPECIMENS
FIXATIVES
246
Cervical / vaginal smears
95 % Ethanol and Ether
95 % Ethanol
247
Sputum and bronchopulmonary
specimens
95 % Ethyl alcohol / Cough directly on a
container with Brasil's fixative
248
Pleural and peritoneal fluids
Brasil's fixative
249
Gastric specimens
95 % Ethanol
250
CELLS FOUND IN VAGINAL
SMEARS
251
1. Mature Superficial
cells
Exhibits true acidophilia ( under estrogen
influence )
It has darken and shrunken nuclei ( pyknotic )
One must differentiate true acidophilia from
pseudoacidophilia which occurs in the
following conditions :
1. Infection
2 .
3 .
4 .
Chemicals
Smear drying before fixation
Vaginal prolapse and drying
252
2. Intermediate cells
-
Medium - sized
Basophilic Cytoplasm with vacuoles
Elongated or polyhedral cells .
It may look like a boat at times and it is called
as Navicular cells
O Found in the latter half of menstrual
cycle , during pregnancy and menopause
O Under progesterone control
253
3. Pregnancy cells
-
-
Oval , round or boat - shaped
Nucleus is eccentric
Has basophilic and translucent cytoplasm
Double - walled boundary with a deep blue
stain at the cytoplasmic periphery
254
4. Parabasal Cells
Parabasal cells
- Round to oval in shape
Smaller than Intermediate cells
Normally found from 2 weeks of age to
puberty , during abortions , after menopause
and after childbirth .
255
5. Endometrial cells
Similar to parabasal cells in appearance
Occurs in groups of 3 or more
Slightly cylindrical with a less basophilic
cytoplasm
Found 1-4 days after menstruation
256
6. Endocervical
glandular cells
- Honeycomb in appearance
With pale blue / gray cytoplasm that is finely
vacuolated
The nuclei have finely granular chromatin
257
7. Basal cells
Basal cells
-
Oval , small and round - shaped
With very large nuclei more than half of the
cell in size
Found before puberty and after menopause
Its cytoplasm is strongly basophilic
