Basic and Advanced Laboratory Techniques in Histopathology and Cytology
Basic and Advanced Laboratory Techniques in Histopathology and Cytology
Basic and Advanced Laboratory Techniques in Histopathology and Cytology
Laboratory Techniques
in Histopathology
and Cytology
Pranab Dey
123
Basic and Advanced Laboratory
Techniques in Histopathology
and Cytology
Pranab Dey
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
part of Springer Nature.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore
189721, Singapore
Dedicated to
Shree Shree Satyananda Giri,
Rini and Madhumanti
Preface
vii
Acknowledgements
I wish to express my thanks to Dr. Naren Aggarwal and Ms. Jagjeet Kaur
Saini of Springer Nature who encouraged me in every stage in this work.
I am thankful to Dr. Suvradeep Mitra who gave me his valuable sugges-
tions at the time of the preparation of this manuscript. He also provided me
many microphotographs. My sincere thanks to Dr. Charan Singh Rayat,
Lecturer in Histopathology, who shared his vast experience in laboratory
technology with me. I am grateful to my technical staffs who exchanged their
views and opinions with me.
My wife Rini and daughter Madhumanti constantly encouraged me during
the writing of this book. They are my source of inspiration.
Lastly I wish to express my gratitude to God because without His blessing
nothing can be done.
ix
Contents
xi
xii Contents
xxi
Abbreviations
ACEP 3Aminopropyltriethoxysilane
APAAP Alkaline phosphatase–antialkaline phosphatase
APC Allophycocyanin
Ab Antibody
AR Antigen retrieval
Acgh Array based CGH
CEA Carcinoembryonic antigen
CEP Chromosome enumeration probe
CI Colour index
CGH Comparative genomic hybridization
CT Computerized tomography
CFM Confocal microscopy
CLM Conventional light microscopy
CP Conventional preparation
CYM Cyan, yellow, and magenta
CK Cytokeratin
DNA Deoxyribonucleic acid
DSRT Desmoplastic small round cell tumor
ddNTP Dideoxynucleotides phosphates
DIA Digital image analysis
EM Electron microscope
EUS-FNAC Endoscopic ultrasound guided FNAC
EA Eosin Azure
EMA Epithelial membrane antigen
ER Estrogen receptors
EDTA Ethylenediaminetetraacetic acid
EWS Ewing’s sarcoma
FOV Field of view
FNAC Fine needle aspiration cytology
FNS Fine needle sampling
FCI Flow cytometric immunophenotyping
FCM Flow cytometry
FITC Fluorescein Iso-thiocyanate
FRAP Fluorescence recovery after photobleaching
FISH Fluorescent in situ hybridization
FPGS FocalPoint GS Imaging System
FFPE Formalin fixed paraffin embedded section
xxiii
xxiv Abbreviations
• To preserve the tissue nearest to its living state 1.4 Tissue Changes in Fixation
• To prevent any change in shape and size of the
tissue at the time of processing The following changes may occur in tissue due to
• To prevent any autolysis fixation (Box 1.1):
• To make the tissue firm to hard
• To prevent any bacterial growth in the tissue 1. Volume changes: Fixatives may change the
• To make it possible to have clear stain volume of the cells. Some fixatives such as
• To have better optical quality of the cells osmium tetroxide cause cell swelling. The
exact mechanism of the change in volume is
not properly understood. However the vol-
1.3 Ideal Fixative ume change may be due to (a) altered mem-
brane permeability, (b) inhibition of the
An ideal fixative should have the following quali- enzymes responsible for respiration and (c)
ties [1]: change of transport Na+ ions. Formaldehyde
The spraying over the smear should be smooth Freeze-drying technique is useful mainly to
and steady, and the optimum distance of study the soluble material and very small
10–12 inches should be maintained between the molecules.
nozzle of the spray and the smear. The spray fixa-
tive usually consists of alcohol and wax. Advantages:
Therefore, this wax should be removed before the
staining procedure. • Excellent for enzyme study
• No change of proteins
3. Vapour fixation: In this type of fixation, the • No shrinkage of tissue
vapour of chemical is used to fix either a • Preservation of glycogen
smear or tissue section. The commonly used
chemicals for vapour fixation are formalde- 6. Microwave fixation (Box 1.2)
hyde, osmium tetroxide, glutaraldehyde and
ethyl alcohol. The vapour converts the soluble Basic principle: Microwave is a type of elec-
material to insoluble material, and these mate- tromagnetic wave with frequencies between
rials are retained when the smear comes in 300 MHz and 300 GHz, and wavelength varies
contact with liquid solution. from centimetre to nanometre. Scientific and
4. Perfusion fixation: This is mainly used in medical microwave ovens operate with a fre-
research purpose. In this technique the fixative quency of 2.45 GHz and 0.915 GHz, respectively.
solution is infused in the arterial system of the The electromagnetic field is created by the micro-
animal, and the whole animal is fixed. The wave, and the dipolar molecules such as water
organ such as the brain or spinal cord can also rapidly oscillate in this electromagnetic field.
be fixed by perfusion fixation. This rapid kinetic motion of these molecules gen-
5. Freeze-drying: In this technique the tissue is erates uniform heat. The generated heat acceler-
cut into thin sections and then rapidly frozen ates the fixation and also other steps of tissue
into a very low temperature. Subsequently the processing. The most important characteristic of
ice within the tissue is removed with the help microwave heat generation is homogeneous
of vacuum chamber in higher temperature increase of temperature within the tissue, and
(−30 °C). every part of the tissue is heated.
6 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives
thereby disruption of the tertiary structure of methylene bridge. This preliminary reaction of
protein. However, the secondary structure of hydroxymethyl side chain is the primary reac-
the protein is maintained. Ethanol is relatively tion, and the subsequent intermolecular and intra-
stronger dehydrating agent than methanol. molecular cross-linking of the molecules occurs
The ethanol and methanol start work from as a slow-growing process. This ultimately pro-
60–80% concentration, respectively. The duces an insoluble product. The formalin can be
dehydrating fixative has two disadvantages: removed from tissue by prolonged washing.
–– Shrinkage of the cells However, once methylene bridge is formed in the
–– Removal of the soluble substances from the tissue, the reaction is stable, and it is difficult to
tissue remove formalin from the tissue. Formaldehyde
• Cross-linking fixatives: also reacts with the nucleic acid by reacting with
Formaldehyde: Formaldehyde in aqueous the amino group of nucleotides.
solution combines with water to form methy- Glutaraldehyde: It has two aldehyde groups
lene hydrate, a methylene glycol: that are separated by three methylene bridges
(Fig. 1.2). The aldehyde group of glutaraldehyde
CH 2 O + H 2 O = OHCH 2 OH reacts with amino group of the protein predomi-
nantly lysine. When one aldehyde group reacts
In a long-standing position, this methylene with the amino group, the other free aldehyde
glycol may further react with water molecules group may help to cross-link. Glutaraldehyde
and form a polymer known as polyoxymethylene rapidly and irreversibly cross-links the protein.
glycol. This again depolymerized in methylene The penetration of glutaraldehyde is slower than
glycol in a neutral buffer system. Formaldehyde formaldehyde.
reacts with various side chain of the protein and Osmium tetroxide: Osmium tetroxide (OsO4) is
forms hydroxymethyl side group (Fig. 1.1). mainly used as a fixative in electron microscopy. It
These compounds are highly reactive and is used alone or as a combination with other agent.
subsequently cross-linking occurs by forming a
O H H H O
C C C C C
H
H OH
H H H H H
H2O
O Glutaraldehyde
C C
H H OH O H H H O
Formaldehyde Methylene glycol
R1-NH2 + C C C C C + R2-NH2
H H H H H Amino
Amino
H group
group
R–H+ C O R – CH2OH
Bound hydroxymethy group
H
H H H H H
R – CH2OH + R – H = R – CH2 – R + H2O
R1 N C C C C C N R2
Metyhylene bridge
H H H
Fig. 1.1 Schematic diagram showing the mechanism of Cross liking with
formalin fixation. Formaldehyde reacts with the side amino group
chain of the protein and forms hydroxymethyl side group.
Later on these highly reactive substances form cross- Fig. 1.2 Schematic diagram showing the mechanism of
linking and methylene bridges are formed. This is a stable glutaraldehyde fixation. The aldehyde group of glutaral-
reaction, and simple washing cannot remove formalin in dehyde reacts with amino group of the protein. Rapid and
this stage irreversibly cross-linking of the protein takes place
8 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives
Fig. 1.3 Schematic O O
diagram showing the
mechanism of osmium Os
tetroxide fixation.
O O
Osmium tetroxide reacts
with two unsaturated Osmium tetroxide
carbon atom of the lipids
and cross-links with
them O O O
HC = HC O
+
Os Os
HC HC O
O O O
Hexavalent osmium
O
HC O + H2O = HCOH + OsO3
Os
HC O
HCOH
O Osmium trioxide
diol
Osmium tetroxide
O O
HC + + CH HC O O CH
Os = Os
HC CH HC O O CH
Unsaturated O O
Unsaturated Cross linking
carbon atom of
carbon atom of
lipid
lipid
acid is converted to NH3, and the reaction enzymes are better preserved in lower tem-
between aldehyde groups of the fixative is perature, and for enzyme histochemistry
reduced. Usually buffer solution is added to 0–4 °C is suitable.
maintain pH of the fixative. The commonly 3 . Duration of fixation: The depth of penetration
used buffers in the fixatives are phosphate, of fixative is directly proportional to the
bicarbonate, Tris and acetate. The buffers square root of time of fixation. The diffusibil-
should be chosen in such a way that they ity of different fixatives may also vary:
should not react with the fixative.
2 . Temperature: Room temperature is alright for D=k T
tissue fixation, and there is no difference of
cell morphology from 0 to 45 °C. However, D = depth of penetration
the fixation time may be reduced in higher T = Time duration
temperature (60–65 °C). At higher tempera- k = Coefficient of diffusion of the fixative
ture the vibration and movement of the mole-
cules are increased. This increases the rate of The penetration rate of formalin solution is
penetration of the fixative within the tissue 1 mm/h. The presence of blood may hamper the
and accelerates the process of fixation. In penetration of the fixative. Therefore it is prefer-
case of very high temperature, the antigen able to wash the tissue specimen thoroughly
within the tissue may be destroyed. The before putting it in fixative. The tissue should be
10 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives
sectioned as 3–5 mm. Overall formalin fixes tis- Volume of formalin: For proper fixation the
sue within 24 h. Prolonged fixation may cause specimen should be sliced in 5 mm apart, and the
loss of lipid and protein and significant reduction amount of formalin should be 20 times the vol-
of the enzymatic activity of the cell. This may ume of tissue. The specimen should be com-
also cause hardening of tissue. pletely immersed in formalin and should not be
in direct contact with the container. There should
4. Osmolarity of the fixative solution: Osmolality be a formalin soaked clothes in between the con-
of the fixative has considerable effect on fixa- tainer and the tissue.
tion. Hypertonic fixative solution causes Removal of formalin from the tissue: As the cross-
shrinkage of the cell, whereas hypotonic fixa- linking of the amino acids and proteins is a slow pro-
tive solution induces swelling of the cells. cess, so if the tissue is washed for 24 h in water, then
Electrolytes (0.9% NaCl) or sucrose may be 50% of formalin from the tissue is removed.
added in the fixative to maintain the osmolar- Precaution: Formaldehyde is irritant to the
ity of the fixative. Mildly hypertonic fixative eye and skin and toxic for inhalation. It is a carci-
(400–450 mOsm) is preferable for routine use nogenic element.
in laboratory.
5. Concentration: Very low concentration of fixa- Advantages:
tive prolongs the time of fixation, and higher
concentration causes rapid fixation. However, • The penetration rate of formalin is high.
higher concentration of fixative may cause tissue • Cell morphology well preserved in formalin.
hardening, tissue shrinkage and artefactual • Cheap.
changes. Mildly lower concentration of fixative • Stable.
with neutral pH is needed for proper fixation. • Easy to make the solution.
Optimal concentration of formaldehyde is 10%. • Formalin is effective fixation for routine labo-
6. Agitation: Agitation increases the rate of pen- ratory staining of the tissue.
etration and therefore rapidity of fixation.
Optimum agitation is needed as slow agitation Disadvantages;
may have no effect of fixation, whereas rapid
agitation may have detrimental effect on deli- 1. Slow fixation.
cate tissue. 2. Formalin reaction with the tissue is reversible,
and it can be removed by washing.
3.
Formalin fails to preserve acid
1.9 ommonly Used Fixatives
C mucopolysaccharides.
in the Laboratory 4. Highly vascular tissue may have dark-brown
granules (artefact)
1.9.1 Formaldehyde 5. Exposure to the skin may cause dermatitis.
6. Chronic inhalation may cause bronchitis.
Pure formaldehyde vapour dissolved in the
water is available as formaldehyde in 37–40%
concentration. This is also known as formalin 1.9.2 Preparation of Different
and is considered as 100% formaldehyde. In Formalin Solution
laboratory 10% of this formalin is used to make
neutral buffered formalin for routine labora- A. 10% neutral buffered formalin:
tory fixative (Box 1.4). • Formaldehyde, 40%: 100.0 ml
Rate of penetration: Formalin penetrates • Distilled water: 900.0 ml
approximately 1 mm/h and usually 24 h is needed • Sodium dihydrogen phosphate: 4.0 g
for fixation of a 1 cm3 tissue. • Disodium hydrogen phosphate: 6.5 g
1.9 Commonly Used Fixatives in the Laboratory 11
Advantages:
Disadvantages:
1. Slow fixation.
2. Formalin reaction is reversible, and it can be removed by washing.
3. Fails to preserve acid mucopolysaccharides.
4. Not good for staining of fat and enzymes.
5. Highly vascular tissue may have dark-brown granules.
6. Exposure to the skin may cause dermatitis.
7. Chronic inhalation may cause bronchitis.
Disadvantages: Preparation
Solution A
1. It produces yellow stain to the tissue.
Distilled water: 250
Removal of yellow colour: Potassium dichromate: 6.3 g
Mercuric chloride: 12.5 g
1. The tissue should be washed thoroughly in Sodium sulphate: 2.5 g
70% ethanol. Solution B
2. This yellow colour can be removed by dipping Thirty-seven percent formaldehyde solution
the tissue in lithium carbonate in 70% Just before use mix 95 ml of Solution A with 5 ml
alcohol. of Solution B.
Table 1.3 Choice of fixative based on technique Table 1.4 Fixative of choice according to tissue
C. Fuzzy Staining
Box 1.5 Fixation Artefact
Appearance: Here the nuclear and cytoplas-
• Formalin pigment: Insoluble brownish-
mic details are obscured and the section looks
black granular retractile birefringent
fuzzy or hazy.
pigment due to reaction of formalin with
Cause: Improper fixation either due to insuf-
haemoglobin derivatives.
ficient fixative or too little time in fixative.
• Mercury pigments: Dark-brown irregu-
lar deposit.
D. Prolonged fixation: Prolonged fixation cause
• Fuzzy staining: Due to improper
shrinkage of the tissue followed by separa-
fixation.
tion. The tissue may show holes or empty
• Prolonged fixation: Shrinkage of the tis-
spaces within it (Fig. 1.6).
sue causes tissue separation and empty
E. Dichromate deposit: If the tissue is not prop-
spaces.
erly washed after dichromate fixation, then
• Dichromate deposit: This deposit may
the chromium salt may form. This chrome
occur after dichromate fixation if the tis-
salt reacts with alcohol, and insoluble yellow-
sue is not washed properly.
brown precipitate may appear.
16 1 Fixation of Histology Samples: Principles, Methods and Types of Fixatives
Fig. 1.5 Microphotograph
shows brownish-black
granular formalin pigment.
This is refractile
birefringent pigment
(haematoxylin and eosin
stain ×400)
Fig. 1.6 Tissue
separation due to
prolonged fixation. The
tissue shows holes and
empty spaces within it
(haematoxylin and eosin
stain ×200)
References 17
to high concentration of dehydration fluid. The • Increases the life span of alcohol
tissue should be kept in the dehydration fluid for • Better dehydration
optimal time because too much time in the dehy- • Good indicator to change alcohol
drating fluid may cause the tissue hard and brit-
tle. Too little time in dehydration fluid may be Methylated Spirit It is also known as denatured
insufficient for removal of free water molecule. alcohol. Methylated spirit contains 99% ethanol
Thin 2–3 mm tissue needs less time in dehydra- and 1% methanol or isopropyl alcohol.
tion fluid than thick 5 mm tissue.
Methanol Methanol is a clear, colourless, vola-
tile and inflammable liquid. It can be used as a
2.3 Individual Dehydrating substitute of ethanol, but it is rarely used in labo-
Agent ratory because of its volatility and high cost.
Toluene It has almost similar properties as that 2.4.2 Other Clear Agents
of xylene. However it does not make the tissue
hard even after prolonged exposure, and its action Esters The different esters are amyl nitrate,
is slightly slower than xylene. Toluene is also methyl salicylate and methyl benzoate. These are
flammable and toxic. less toxic and may be used in manual processing.
They do not cause tissue hardening even under
Chloroform It is highly volatile, non-inflammable, prolonged exposure.
expensive and toxic agent. The penetrating power of
chloroform is slower than xylene. However, it does Cedarwood Oil This is an expensive rapid
not cause any tissue shrinkage and is mainly used in clearing agent and mainly used in clearing dense
the uterus, muscle and other dense tissue. Presently tissue.
chloroform is rarely used in laboratory.
Limonene This is a clear liquid. It does not cause
Table 2.2 compares the different clearing any tissue hardening. However, the removal of lim-
agents commonly used in laboratories. onene from the tissue by paraffin wax is difficult.
24 2 Processing of Tissue in Histopathology Laboratory
2.5 Infiltration and Embedding 1. Size of tissue: Thicker large tissue takes more
time to impregnate with the embedding medium.
This is the next step after clearing. The clearing It also contains more clearing agent to remove.
agent within the tissue is removed by the process of 2. Type of tissue: Hard tissue such as bone and
diffusion. The tissue space is now infiltrated with cartilage takes more time for embedding than
the embedding media. Usually molten wax is used soft tissue.
as the embedding medium. After cooling in room 3. The type of clearing agent: Certain clearing
temperature, the molten wax is solidified to provide agents are easy to remove than others. Such as
support for cutting into thin section (Box 2.4). xylene and toluene are easy to remove than
Ideal impregnating medium: An ideal impreg- cedarwood oil.
nating medium should have following qualities: 4. Type of processing: Vacuum embedding
enhances impregnation.
• Miscible with clearing agent
• Liquid in higher temperature (30–60 °C) and
solid in room temperature 2.5.1 Different Impregnating
• Homogenous and stable Medium
• Non-toxic and cheap
• Transparent 2.5.1.1 Paraffin Wax
• Fit for sectioning the tissue Paraffin wax is a type of hydrocarbon and is produced
as by-product during refining of crude petroleum.
The time duration and the number of changes This is the most popular universally accepted embed-
required for the impregnation in tissue depends ding medium for tissue processing. This is non-toxic
on: and inexpensive medium. The melting point of paraf-
fin wax varies from 39 °C to 70 °C. The wax is sold a handful number of tissue. Automated tissue
according to its melting point. Paraffin wax with low processor is widely used in laboratories.
melting point is soft in room temperature, whereas Automated tissue processor: The basic principle
paraffin wax of higher melting point is much harder in of tissue processor is to transfer the tissue in differ-
consistency. Therefore, it is necessary to have paraffin ent fluid for a specified time in a desired environ-
wax that has suitable melting point to get good ribbon ment. There are two types of tissue processor:
of tissue. In this Indian subcontinent, the paraffin wax
with melting point around 60 °C is the most suitable 1 . Tissue transfer processor
for laboratory use. Total 3–4 h’ time in paraffin wax is 2. Fluid transfer processor
sufficient for impregnation of tissue by wax. 1. Tissue transfer processor (Fig. 2.2): In this
system the bucket of tissue is transferred from
Advantages of paraffin wax: one carousel to other after a specified time.
There are several containers with reagents.
• Tissue block can be stored for long duration. Tissue remains in a basket with 30–100 cas-
• Non-toxic. settes. The basket containing the tissue is sub-
• Cheap. merged in the specific container for a particular
• Safe. time and then transferred to the next container
automatically. A gentle agitation is created by
Disadvantages of paraffin wax: vertical oscillation or by rotatory movement
of the tissue basket. The time schedule and
• It may cause tissue shrinkage and hardening in transfer of tissue in each container are deter-
case of prolonged impregnation. mined by a microprocessor.
• Paraffin wax takes long duration for the 2. Fluid transfer processor (Fig. 2.3): This is a
impregnation of the bone and eye. completely closed processor. Here the tissue
is kept in the container, and the container is
2.5.1.2 Additives and Modification periodically filled with particular fluid. After
of Paraffin Wax a certain period, the fluid is pumped out from
To alter the physical characteristics of paraffin the container containing the tissue. It is again
wax, the following modifications may be done: filled with the fluid required for the next step.
In this processor each step can be customized
1. To increase hardness: addition of stearic acid for vacuum, temperature, and time duration.
2. Reduction of melting point: addition of
phenanthrene
3. Improving adhesiveness with tissue and wax:
addition of 0.5% of ceresin
Tissue processing can be done by simple manu- Fig. 2.2 Tissue transfer automatic tissue processor. Here
ally or by automated processor. Manual tissue the whole bucket containing tissue is automatically trans-
processing is done only in a small laboratory with ferred to the next fluid station
26 2 Processing of Tissue in Histopathology Laboratory
Alcohol (graded)
IHr
Dehydration
IHr
Paraffin
Embedding
100%
IHr
IHr
Paraffin Clearing
Fig. 2.3 Fluid transfer advanced automatic tissue proces- 100%
sor. Here the fluid itself is changed automatically, and the Xylene
bucket remains static Xylene Xylene
IHr IHr
Advantages:
IHr
1. Vacuum pressure makes the system faster and IHr IHr
efficient.
Fig. 2.4 Schematic diagram showing the time schedule
2. In this closed system, there is no chance for
of overnight processing
tissue drying.
90% ethanol: 1 h
Absolute alcohol: 1 h
2.7 verall Precautions of Tissue
O Absolute alcohol: 1 h
Processing Absolute alcohol: 1 h
Xylene/toluene: 1 h
1. The bulk of the tissue should be optimum for Xylene/toluene: 1 h
adequate penetration of fluid. Xylene/toluene: 1 h
2. The amount of fluid should be adequate and Paraffin wax: 1 h
the fluid level should be always higher than Paraffin wax: 1 h
the tissue level. Paraffin wax: 1 h
3. The tissue basket and cassettes should be
clean and any spillage of wax should be
cleaned. 2.7.2 Manual Tissue Processor
4. The temperature of the infiltrating medium
should be optimum, and it is preferable to It is rarely used in routine laboratory. The advan-
keep the temperature 3–4 °C above the melt- tage of manual processing are:
ing point.
5. There should be a proper record of the change 1. Small number of samples can be processed in
of fluid, number of tissues processed, etc. a small laboratory.
2. Careful monitoring in each step is possible.
3. In case of emergency when the automated tis-
2.7.1 T
ime Schedule for Overnight sue processor is not working, one can take the
Processing (Fig. 2.4) help of the manual processing.
4. In case of manual processing, it is possible to
50% ethanol: 1 h select the reagents of choice with flexibility in
70% ethanol: 1 h time duration.
References 27
The major disadvantages of manual process- The microwave processing may be used for all
ing include inconvenient for processing and time the steps of processing.
taken procedure. Tissue processing for electron microscopy:
See Chap. 26.
Troubleshooting in processing is highlighted
2.7.3 Microwave Processing in Table 2.3.
After processing the tissue, the next step is 3.1 Embedding Medium
embedding of the tissue to make the block. In the
embedding process, the tissue is surrounded in a (a) Paraffin wax: As described in the previous
molten medium by using a mould. Subsequently chapter, paraffin wax is a solid polycrystal-
this medium is solidified to make a block for cut- line hydrocarbon. The paraffin wax is sold in
ting thin section of tissue. the market with different melting point.
Aims of embedding: Embedding medium has Paraffin wax with melting points ranging
three important functions: from 56 to 62 °C is used in our laboratory.
Paraffin wax is cheaper and easy to use. Little
1 . To give support of the tissue supervision is needed to make block by it.
2. To prevent distortion of the tissue during
(b) Epoxy resin: Epoxy resin is mainly used in
cutting electron microscopy as it provides better res-
3. To preserve the tissue for archival use olution and greater details of tissue.
(c) Acrylic medium: Methacrylate monomer is
The choice of the embedding medium: Various miscible with ethanol. In the presence of cata-
media are used for embedding such as paraffin lyst (benzoyl peroxide 2%), methacrylate
wax, epoxy resin, methacrylate, carbowax, etc. monomer is polymerized and provides a hard
Paraffin wax is the most commonly used embed- and clear block. Methacrylate monomer is
ding medium. The choice of the embedding available in the market along with hydroqui-
medium depends on the following factors: none which should be removed by treating with
weak alkali solution followed by thoroughly
1. Type of tissue: The density of the tissue and washing with water. The presence of water may
the embedding medium should be close other- lead to small bubbles within the block.
wise tissue may not be sectioned properly, and (d) Agar gel: Agar gel helps in cohesion of friable
tissue will be deformed. and fragmented tissue particularly in cytology
2. Type of microtome sample and also endometrial curetting and
3. Type of microscope small endoscopic biopsies. It does not provide
good support of the tissue for section cutting.
The basic technique of embedding is the same Agar-paraffin wax double embedding is more
irrespective of the embedding medium. suitable technique than agar alone.
L shaped metal
plate is flat and well-polished that helps to remove liq-
uid paraffin. The mould can be covered by a plas-
tic ring.
Forceps warmer
The mold is covered with Unique number is put in The moulds are put on the
Tissue is pressed on mould
peripheral plastic ring the Plastic ring cold plate
Fig. 3.5 Illustrated view of whole embedding process. At molten paraffin. The mould is now covered with the
first the Tissue-Tek system is put on. The tissue is taken peripheral plastic ring. A unique number is placed in the
out from the processing bucket. The molten paraffin is plastic ring and the mould is now kept on the cold plate to
poured in the metal mould. The tissue is embedded with make it firm
the help of forceps. The tissue is pressed to keep in the
32 3 Embedding of Tissue in Histopathology
Tissue-Tek System II The basic steps of Tissue- 2 . Tissue with epithelial surface should be placed
Tek II are similar as described above. In this sys- vertically and right angle to the surface so that
tem instead of metallic mould, we use plastic we can get all the layers.
mould to hold the liquid paraffin. 3. Multiple section of tissue such as endometrial
curetting should be placed all in central position.
4. Linear long tissue should be placed diagonally.
3.4 Tissue Orientation 5. Muscle biopsy should be placed both in longi-
and Embedding tudinal and transverse plane.
6. Long membranous tissue such as amniotic
The correct orientation of the tissue is very membrane should make as Swiss roll.
important for proper cutting and microscopic
examination. Tissue is usually placed as flat on
the central part of the mould. It should be ori- 3.5 Tissue Marking [2]
ented in such a way so that cutting is easy by
knife of the microtome. Some of the tissue The tissue marking by ink is needed for the fol-
needs special care as described below lowing purposes:
(Fig. 3.6):
1. To identify the resection plane or outer margin
1. The tubular tissue (fallopian tube, vas differ- of the tissue
ence, artery, etc.) should be placed in such a 2. To help in embedding the tissue
manner so that we get a transverse section 3. Any area of interest to identify such as the area
with all the layers. of transitional zone in cone biopsy of cervix
Fig. 3.6 Illustrated
view of embedding of
different tissue samples
References 33
The tissue markers should have the following Table 3.1 Troubleshooting in tissue embedding
characteristics features: Problem Cause Remedies
Fraction of the Tissues are Give pressure after
• The marker substance should not be dissolved embedded embedded in embedding the
in fixative and tissue processing agents. tissues are different level tissue in the
cutting molten wax in
• The marker should not penetrate the deeper mould
tissue. Tiny fragments Tissue is – Clean the
• It should be recognizable in the stained section of tissue is seen carried over forceps every time
both microscopically and macroscopically. in the by forceps after embedding
subsequent – Deal with only
The common tissue markers: The common tis- blocks one tissue at a
sue markers include: time, and so open
• India ink: This is the most commonly used only one cassette
marker in the routine surgical pathology labo- at a time
ratory. It takes 15 min time to mark the Epithelium not Wrong Please mark the
properly seen orientation tissue by ink so
tissue. that the embedding
• Silver nitrate: This is also a good marker. It upper surface can
produces brown-black colour. be identified
• Rose Bengal: For surgical margin stain, 1% Tissue is fallen Air bubbles Properly embed
out during are entrapped the tissue in the
Rose Bengal dye is used. It stains within 5 min
microtomy around the molten wax
and provides pink stain. tissue
• Application of ink:
• Clean the area and dry the tissue with bloating
paper. Completely dry the tissue. References
• Apply the dye by a cotton swab.
• Allow some time to dry it. 1. Mccormick JB. Improved tissue-embedding method
• Put fixer over the dye. Usually 3% acetic acid for paraffin and carbowax, using Tissue-Tek system.
Tech Bull Regist Med Technol. 1959;29(1):15–6.
or 50% white vinegar is used as fixer. 2. Dimenstein IB. Grossing biopsies: an introduction to
• Dry the specimen with a sponge. general principles and techniques. Ann Diagn Pathol.
• Process now. 2009;13(2):106–13.
• Formic acid
• Washing: The fixed tissue should be washed • Trichloroacetic acid
thoroughly before decalcification.
• Choice of decalcifying agent: Suitable choice Strong Acid The strong acids are used in 5–10%
of the decalcifying agent is required. concentration. They are rapid in action. However,
• Volume: Optimum volume of the decalcifying careful attention is needed to prevent tissue dam-
agent is a prerequisite for proper decalcification. age. Neutralizer is also used to prevent any tissue
• End point detection: The end point of the decal- distortion.
cification should be determined correctly.
Aqueous Nitric Acid This is rapid in action. It
does not impair staining if the end point is not
4.1.1 F
actors Controlling the Rate crossed.
of Decalcification
Preparation:
• Concentration: The increased concentration
Nitric acid 5 ml
of the decalcifying agent increases the rate of Distilled water 100 ml
decalcification.
• Temperature: Increased temperature fastens
Advantages:
the decalcification rate.
• Density of bone: Hard bone takes longer time
1 . Rapid in action
to be decalcified.
2. Good nuclear stain
• Agitation: Mild agitation of the decalcifying
solution increases the rate.
Precaution: Nitric acid may give yellow
• Thickness of tissue: Thinner tissue is quickly
colour to the tissue that can be removed by urea.
decalcified.
Nitric acid formaldehyde (10%)
agents in the routine laboratory decalcification 2. Various other laboratory tests can be done on
(Box 4.2). It binds with calcium of the hydroxy- the tissue such as immunohistochemistry, flu-
apatite crystals and forms a non-ionized soluble orescent in situ hybridization technique, etc.
complex. The action of EDTA is slow and gentle, 3. It is very good for bone marrow trephine
and it may take several weeks to remove calcium biopsy as glycogen is preserved in the
from the tissue. Therefore EDTA is not a suitable tissue.
decalcification agent for dense bone or urgent
removal of calcium. The main advantage of Disadvantages:
EDTA is the preservation of morphology and to
maintain the tissue for various other techniques 1 . Very slow process.
for research purpose. The action of EDTA is pH 2. Maintenance of pH around 7 is necessary.
dependant and it works best in pH 7–7.6. 3. Thin tissue is needed.
EDTA solution
EDTA 5.5 g
Formalin 100 ml 4.3.1 Other Procedures
Distilled water 900 ml of Decalcification
5.1 Introduction
5.2 Microtomes
• Rotary microtome
• Rocking microtome
• Base sledge microtome Fig. 5.1 Semiautomated rotary microtome
• Sliding microtome
• Cryomicrotome
• Ultramicrotome tory handle attached with the microtome. In
• Laser microtome each 360° rotation of the wheel handle, the
block moves down followed by up, and the
(a) Rotary microtome (Fig. 5.1): This is the most tissue is cut as thin ribbon. This microtome
commonly used microtome in routine labo- has the option to be semiautomated or auto-
ratory. The cutting blade is kept in horizon- mated with the adjustment and control of
tal position, and the block containing tissue the movement of the block and angle of the
moves up and down with the help of rota- knife.
Advantages: Disadvantages:
1. Good-quality 2–3-μm-thin section is 1. Difficult to get thin section.
possible. 2. Large slides are required.
2. Heavy and stable automated rotary
(d) Sliding microtome: In this microtome the
microtome reduces health hazard and knife is static, and the block moves horizon-
gives the best-quality section. tally over the knife.
3. Good tissue ribbon production.
4. Easy-to-cut various types of tissue: firm, Advantages:
fragile, small biopsy, etc. 1. Large sections can be cut.
2. Mainly used for celloidin-embedded tissue.
Disadvantages: 3. Simpler design and easy maintenance.
1. Expensive. 4. Brain sections can be cut better by this
2. Unsuitable to cut large block. type of microtome.
3. Knife faces up and so may be dangerous
to the technical staff. Disadvantages:
(b) Rocking microtome: The rocking microtome is 1. The knife may glide in case of hard tis-
also known as Cambridge rocking microtome. sue and may jump.
The word “rocking” is used as there is a rock- 2. Long knives are difficult to sharpen.
ing action of the microtome like arm move- (e) Cryomicrotome: This type of microtome is
ment. In this type of microtome, the knife is used for the cutting tissue for frozen sample
static, and the block of tissue moves in a rock- (Fig. 5.2). The sample is made hard in liquid
ing motion (arc-like movement of the block). nitrogen and then cut by the microtome in the
This is one of the oldest designs of the micro- chamber that contains liquid nitrogen.
tome. The microtome can cut thin section with
ribbons and is ideal for serial section. The sec- Advantages:
tions are slightly curved in this microtome. 1. To get rapid section for rapid diagnosis
2. To study nerve biopsy
Advantages: 3. To study enzyme histochemistry
1. Thin section
2. Easy to operate Disadvantages:
3. Low-cost instrument and reliable 1. It needs continuous supervision to main-
tain the temperature.
Disadvantages: 2. Freezing artefact is often seen.
1. Tissue is curved and the microtome does 3. Very expensive instrument.
not provide flat section. 4. Fixed tissue is very difficult to cut.
2. As the microtome is of light weight so
vibration may occur.
(c) Base sledge microtome: In sledge microtome
the block is fixed in a static position within a
steel carriage. The knife slides to and fro over
the top of the block. This microtome is the
best for large tissue sample or the hard tissue.
The tissue sections are usually thick (more
than 10 μm) in base sledge microtome.
Advantages:
1. Hard tissue can be cut.
2. Large tissue sample can be cut.
3. The best microtome for ophthalmology
and large neuropathology section. Fig. 5.2 Cryomicrotome used in frozen section
5.1 Introduction 43
steep. The tool edge knife is mainly used to cut Perpendicular line
the hard tissue such as decalcified bone. The From surface
of block
knife is difficult to sharpen and is not recom- Rake angle
mended presently.
Knife
Cutting
angle
5.2.2 Disposable Knife Angle of
clearance Upper
Nowadays disposable blade is used in many labo- Block surface
ratories to save time to sharpen. Two types of dis-
posable blades are available: Fig. 5.4 Schematic diagram of angles of knife
several times. Same procedure is followed in the tissue, one can add a few drops of alcohol or
other surface of the knife also. little amount of detergent. This reduces the sur-
face tension of the water and tissue floats
Stropping It helps to polish the cutting edge of smoothly.
the knife that is already sharped by honing and
also to remove any “burr” formed during honing. Blunt Forceps and Camel Hair Brush Blunt
Strop is made of leather and it should be free forceps helps to manipulate the floating tissue
from any dust or grit. section (Fig. 5.6). Camel hair brush is used to
clean the blade.
Automatic Knife Sharpener Presently auto-
matic knife sharpener is available in the market. Slide Rack with Clean Glass Slides The clean
The knife is placed horizontally on the surface of slides are kept in the slide rack. The slides can be
the rotating plate made of glass or copper coated already labelled by diamond pencil or on the
with abrasive agents. frosted side by lead pencil. Alternatively this can
be marked after lifting the tissue section.
5.3.2 Factors Involved in Cutting Adhesive In case of routine section and stain-
ing, no adhesive is required. However, in cer-
1. Temperature: Lowering the temperature facil- tain situations we use cell adhesive material
itates section cutting. such as:
2. Angle of rake: Higher rake angle helps in
smooth flow of ribbons. Lower rake angle is
used for hard tissue.
3. Consistency of tissue: Soft tissue is cut at a
slow rate than the hard tissue.
Cooling
Trimming the block
of tissue by ice Cutting the tissue and
making ribbons of tissue
Fig. 5.7 Different steps of section cutting are highlighted and the tissue ribbon is placed with the help of brush on the
here. At first the tissue is trimmed. Then the block is cooled water bath. Then the tissue is picked up by a putting a glass
in ice. The block is placed in the microtome, and the angle slide perpendicularly in front of it, and then when the tis-
of clearance is adjusted to 5 °C. The tissue is gently cut, sue is touched, the slide is withdrawn vertically
5.4 Sectioning the Paraffin Block 47
3. Cutting proper: The block is fixed in the chuck 4. Floating the ribbon: The ribbon of the tissue
of the microtome. The cutting surface of the is floated in the water bath, and this makes the
block should be parallel to the knife. The tissue flat and removes any wrinkling of the
angle of clearance should be only 2–5° to have tissue. With the help of the forceps, the indi-
good section (Fig. 5.8). The tissue in the block vidual sections are separated from each other.
is cut by gentle, smooth and slow stroke. The As mentioned before, the temperature of the
ribbon-like tissue sections are produced. The water bath should be constantly maintained
tip of the ribbon is held by forceps, and the below the melting point of the paraffin wax. In
end part of the ribbon is removed from the case of temperature variation in the bath, the
knife edge by brush. In case of any difficulty air bubbles may be formed that may rupture
to get the flat section, the cutting surface the tissue.
should be gently warmed by warm water. 5. Picking up the tissue: The slide is placed verti-
cally within the water bath in front of the tissue,
and when the tissue is touched, the slide is
withdrawn vertically from the water. The tissue
pickup process must be gentle and smooth. To
Block
prevent any mix-up, the water bath should be
Tissue
cleaned immediately after cutting each block.
6. Drying the section: The slide containing the
picked-up section is kept in slide rack. The
slides are now kept in hot oven to get dry. The
Angle of clearance
temperature of the oven should be slightly
Knife
more than the melting point of the paraffin.
Fig. 5.8 The schematic figure of the tissue and the posi-
Various problems may occur in tissue section-
tion of knife making the correct angle of clearance which ing (Figs. 5.9, 5.10, 5.11, 5.12 and 5.13) [1].
is 5° These are enumerated below in Table 5.1.
Fig. 5.12 Freezing
artefact of the tissue due
to immediate putting the
tissue from the
refrigerator to formalin.
Note the regular spaces
in between the tissues
due to melting of ice
crystals
Fig. 5.13 Uneven
staining pattern due to
poor deparaffinization of
the tissue
50 5 Tissue Microtomy: Principle and Procedure
Reference
1. Peachey LD. Thin sections. I. A study of section thick-
ness and physical distortion produced during microt-
omy. J Biophys Biochem Cytol. 1958;4(3):233–42.
Frozen Section: Principle
and Procedure 6
Fig. 6.1 Cryostat
machine with its parts
Place to keep the brush and knife holder: Just 6.4 Cryostat Sectioning
in front of the microtome machine, there remains
a small place to keep the brush and knife holder. The process of the cryostat sectioning needs the
Knife or blade: Nowadays, low- or high- following steps.
profile disposable blades are used. The blade
should be proper fixed to the holder to get an even 1. Grossing and cutting the specimen (Box 6.2):
pressure in the whole length. Alternatively Profile The cutting surface of the tissue should be
C steel blade is also used. The angle of the knife smooth. The following steps in grossing of the
is kept in between 5° and 7°. tissue are mandatory for accurate reporting:
Antiroll plate (Fig. 6.2): Just in front of the • Identify the tissue sample of the patient and
knife, there is an antiroll plate that prevents the the requisition form: This is the first and
rolling of the cut tissue. It is usually a glass plate foremost part of the frozen tissue grossing.
6.4 Cryostat Sectioning 53
a b c
d e f
Fig. 6.3 Cryostat processing: (a) mould is covered with cooling chamber, (e) the brush guides the tip of the tissue,
OCT, (b) the tissue is now put on the block, (c) OCT is (f) the tissue section is gently spread over the antiroll plate
flooded over the tissue, (d) the tissue now is put in the and later picked up by touching a glass slide
• 95% ethanol for 10 s. Table 6.2 Optimum temperature for frozen section
• 100% ethanol for 10 s. Optimum
• 100% ethanol for 10 s. Tissue temperature
• Dip in xylene for 20 s. Brain, liver, spleen −7 °C to −10 °C
• Mount by DPX. Rectum, uterus, adrenal, muscle, −12 °C to −15 °C
skin
Heart, lung, intestine, pancreas, −16 °C to −20 °C
ovary, cervix, prostate
6.5.2 Toluidine Blue Stain Bone marrow, breast −20 °C to −25 °C
H
C
C
C
H C C H
H C C H
2[o]
C H
H C C
H C C H
Fig. 7.1 Benzene ring.
Benzene itself is C
colourless. When two H C
atoms in benzene ring
H Active
were replaced by two O
atoms, then the Benzene chromatophore
O
compound quinone group
(C6H4O2) is formed Quinone
which is a chromogen Colourless Chromogen
How Dye Produces Colour The visible light • Chromophore groups: The chromophore
has the range of wavelengths between 400 and group absorbs light and imparts colour to the
650 nm. The white light contains all the seven stain. These groups have many free electrons
colours (VIBGYOR: violet, indigo, blue, green, that absorb the ultraviolet rays of light which
yellow, orange and red) with the wavelength in are not in the visible range.
between 400 and 650 nm. A chromogenic dye • Auxochrome part: This part of the dye helps to
absorbs the light of particular wavelength of the intensify the light. It is an ionizing group that
white light representing a specific colour and also helps to stick the stain with the tissue.
emits the light containing the rest of the colour. The auxochrome group augments more free
Therefore we see the particular colour of the electrons in the chromophore groups. The
dye,such as the dye that absorbs red light will be increased number of electrons in the system
visible as green coloured in the naked eye. helps to absorb light of longer wavelength in
the visible range.
The dye is the most elementary component of
staining of a tissue. In general dye has two com-
ponents: chromophore and auxochrome part 7.1.2 Types of Dye
(Fig. 7.2):
The dye can be classified on the basis of electri-
cal charge (Table 7.1):
(b) Cationic dye or basic dye: Cationic dye car- 7.1.3 Types of Dye Based
ries a positive charge (coloured cation) and on Chemical Structures
move towards cathode in an electrical field. and Chromophore Groups
The cationic dyes are mostly soluble in etha-
nol. These are basic dyes and combine with 1. Azo dye: These dyes contain –N〓N– chro-
tissues that carry negative charge. This nega- mophore group. Majority of the azo dyes are
tively charged tissue combining with basic anionic (acid) dye. Example: orange G and
dye is called as “basophilic” tissue. Congo red
Example: The examples of basic dye are 2. Thiazine dye: Thiazine dye contains –C–
methyl green, ethyl green and Alcian blue. N〓C– and –C–S〓C chromophore group.
(c) Neutral dye: These are the compound dyes Example: toluidine blue and methylene blue
that contain both acid and basic dyes in com- 3. Triphenylmethanes: Triphenylmethanes con-
bination. In aqueous solution, the acid dye tain 〓N– chromophore group. Example:
and basic dye exchange electrons and com- methyl violet, light green and malachite green
bine together to precipitate in the tissue. 4. Azin dye: This group of dye contains C–O〓C
Romanowsky-Giemsa staining is the best and C–N〓C chromophore. Example:
example of the compound dye that undergoes Celestine blue and Nile blue sulphate
electron transfer process. Romanowsky- 5. Diphenylmethanes: They contain –NH chro-
Giemsa contains azure B and eosin Y. The mophore group. Example: auramine
azure B dye is the electron acceptor, and 6. Xanthene dyes: Example—eosin, Rose Bengal
eosin Y is electron donor. and phloxines
Example: Giemsa 7. Oxazine dyes: They contain C–O〓C chromo-
(d) Ligand or chelating dye: Ligand dye is the phore group. Example: cresyl violet and
complex compound that consists of dye and a Celestine blue
metal ion. They are also known as metallo- 8. Acridine dyes: The dyes of this group are
chrome. They are usually weak acids. derived from acridine. Example: acridine
Haematein is the oxidized haematoxylin, and orange
it is used as a combination of aluminium (Al) 9. Anthraquinone dyes: These dyes are derived
ions or iron (Fe) ions. The metal-ion complex from anthraquinone. Example: carminic acid
has surplus charge that increases the solubil-
ity in water and makes the dye insoluble in
alcohol, and therefore dehydration due to 7.2 Mechanisms and Theory
ethanol does not occur during staining. of Staining
Al3+-Haematein It is a type of ligand dye where The staining is the combination of a coloured
the metal aluminium (Al) is combined with hae- substance (dye) with the tissue that retains the
matein. It is used in Harris haematoxylin and dye after washing. The staining is primarily a
Mayer’s haematoxylin. As Al3+-haematein is chemical reaction between the dye and the tis-
insoluble in ethanol, it can be used along with sue. The following chemical reactions are
other anionic dyes, and both dyes are retained involved between the dye and tissue compo-
even after dehydration with alcohol. nents (Box 7.1) [1, 2]:
Electrons
Dipole-dipole interaction
Dipole
Dipole
Fig. 7.4 Schematic diagram of van der Waals force. The dipole. In case of London force, the permanent dipole
positive charge of a permanent dipole interacts with other induces the adjacent atom as induced dipole that further
permanent dipoles. In case of dipole-induced dipole inter- induces a chain of induced dipole and forms a large net-
action, the permanent dipole interacts with induced work of tissue with induced dipole interaction
induced dipole. The induced dipoles further with each other to satisfy the outer shell’s
induce a chain of induced dipole. In this way a required number. The covalent bond is a stron-
large network of tissue may undergo induced ger bond.
dipole interaction. This is known as dispersion Example: periodic acid Schiff’s staining
or London force. for glycogen and Feulgen reaction.
Example: elastin stain by Miller’s stain and 5. Hydrophobic bond: This is a misnomer as in
Congo red stain. standard chemistry, there is no such bonding. It
3. Hydrogen bond: Hydrogen bonding is a weak is probably a type of van der Waals force. When
bond. It is a type of covalent bond that occurs two hydrophobic molecules interact, then
between hydrogen and a strong electronegative London force, the dispersion type of van der
atom commonly O, N or F. Water forms hydro- Waals force, interacts. Therefore instead of
gen bond and so competes with stain- tissue hydrophobic bonding, the better terminology is
bonds. Therefore, hydrogen bonding in dye-tis- probably “hydrophobic interaction” [3].
sue less likely occurs in aqueous solution. Example: staining in aqueous solution and
Example: Best’s carmine dye to stain glycogen. metachromatic staining.
4 . Covalent bond: In case of covalent bond, the 6 . Dye aggregations: Dye molecules may inter-
two electrically neutral atoms share electron act with each other forming dye-dye
62 7 Staining Principle and General Procedure of Staining of the Tissue
interaction. They aggregate in solution and s ubstances. Affinity of the dye to the specific
then penetrate into tissue. The dye-dye aggre- tissue is also influenced by the pH and the
gate increases when the dye concentration is presence of inorganic salt concentration of the
high, the molecular size of the dye is bigger solvent.
and temperature is low. 2. Specimen geometry: Specimen geometry or
topography also influences the staining.
(a) Thick tissue: If the tissue is thick, then the
7.3 Factors Influencing Staining penetration of dye is difficult, and the
central part of the tissue takes poorer
Several factors have influence on staining inten- stain.
sity (Box 7.2): (b) Surface topography: The surface of the
tissue of paraffin section is more even
1 . Dye affinity to the target tissue specimen than cryostat section, and so it takes better
2. Specimen geometry stain.
3. Target concentration (c) Disturbance of microtopography of tis-
4. Rate of reaction sue: The alcohol is a coagulative fixative
5. Rate of stain loss that disturbs the topography of the cell
1. Dye affinity to the target tissue specimen: The and tissue. The shattering effect of alco-
tendency to bind a dye with the target tissue is hol increases the dye penetration rate.
known as dye affinity. The acidic dye such as (d) Inner geometry of tissue: The inner geom-
eosin binds strongly with acidophilic target, etry of the tissue may also influence stain-
that is, cytoplasmic protein. The acidic dye ing such as bone marrow canaliculi which
has very little affinity with basophilic are rapidly stained by Schmorl’s thionine
stain than the adjacent connective tissue.
3. Target concentration: The concentration of
Box 7.2 Factors Influencing Staining the target tissue affects the staining intensity
Intensity as the more the amount of the target tissue, the
• Dye affinity to the target tissue speci- more intense will be the staining.
men: Acid dye binds with positively 4. Rate of reaction: Rate of reaction in the target
charged acidophilic tissue, and basic tissue also influences the staining pattern such
dye binds with negatively charged baso- as in Feulgen reaction, the short reaction time
philic tissue. exposes only a few aldehyde groups produc-
• Specimen geometry: ing weak staining pattern.
–– Thick tissue: Less penetration of 5. Rate of stain loss: Staining pattern is greatly
dye. influenced by rate of stain loss. At times the
–– Surface topography: More even sur- stain loss may be intentional such as differen-
face gives better stain. tiation or destaining. The differentiation often
–– Microtopography of tissue: Alcohol removes the excess stain from the cell and
fixation disturbs microtopography. thus helps to differentiate the organisms.
–– Inner geometry of tissue.
• Target concentration: The more the
amount of the target tissue, the more 7.3.1 Nomenclature Used
intense will be the staining. Regarding Dye
• Rate of reaction: Short reaction time
often decreases stain intensity. Colour Index of Dye A certain dye may be called
• Rate of stain loss: Too much differentia- in different names, or the same name may be given
tion often removes the stain. to many different dyes. Therefore the simple name
of a dye may evoke confusion. To avoid this
7.4 Metachromasia 63
Anionic Dyes Only a few anionic dyes are meta- Factors enhancing metachromasia:
chromatic such as Biebrich scarlet and bromo-
phenol blue. These anionic dyes are weakly • Higher dye concentration
metachromatic. • Low pH
• Decreased temperature
Mechanism of Metachromasia (Fig. 7.5) • Aqueous solution
Glycosaminoglycans of the connective tissue and
epithelial mucins and granules of mast cells are
negatively charged polyanions. The cationic
This causes metachromasia or altered colour,
(positively charged) dye in aqueous solution
such as pyronin Y in the tissue that gives red to
reacts with the polyanions of the tissues. The
orange colour.
binding of the dye molecule with these polyan-
ions of the tissue neutralizes the positively
Various Types of Metachromasia (Fig. 7.6) In
charged dye. The nonpolar aromatic ring of the
relation to thiazine dye, three types of metachro-
dye binds with the other dye by van der Waals
matic change may be seen [5]:
force. The dye-dye aggregation occurs, and
dimer, tetramer and polymer of the dye molecules
• Alpha (orthochromatic): The dye remains in
are formed. Overall dye binding becomes stron-
monomeric form and gives blue colour.
ger due to van der Waals force. The dye absorbs
• Beta (di- and trimeric form): The dye forms
light of shorter wavelength, and the visible colour
dimeric structure and produces purple colour.
of the light emitted from the dye tissue changes.
64 7 Staining Principle and General Procedure of Staining of the Tissue
Water molecule
Dye with
Polyanionic tissue intercalated
water molecule
Dye is neutralized
by poyanioninc tissue
Polyanionic tissue
Dye aggregates
Fig. 7.5 Mechanism of dye aggregation in metachroma- dye aggregates and the absorption of light changes by the
sia. The cationic dye interacts with the polyanionic tissue, aggregated dye-tissue complex
and the bound water of the dye molecule is released. The
Dye
Tissue
Tissue
Tissue
Fig. 7.6 The different
types of metachromasia
are highlighted in this
Polymeric: red colour
schematic diagram Gamma
7.6 Mordant 65
• Gamma (polymeric form): The dye is in poly- (b) Oxidizing agent: The oxidizing agents are
meric form and produces red colour. used to oxidize the dye and make it a colour-
less material such as picric acid, potassium
The colour changes in metachromasia may permanganate, etc.
not be homogenous. At times the anions in the (c) Mordant: Here the dye-mordant complex at
tissue may be widely distributed. In such cases, first binds with the tissue. Subsequently
there may be purple colouration of tissue due to excess mordant is used that attracts the
the mixture of orthochromatic blue and polychro- attached dye in the tissue. The mordant
matic red colour. thereby removes the excess dye from the
tissue.
Factors Influencing Metachromasia The fol- (d) One dye is replaced by other less affinity
lowing factors may influence on metachromasia: dye.
therefore the staining is not altered even after The bench should be cleaned properly with
subsequent treatment of the tissue. arrangement of fume remover. There should be
Mordant may be used in three ways: at least two supplies of running tap water with
sink.
1. Pre-mordanting: The tissue is at first treated
with mordant followed by dye. Stains and Equipment The reagents should
2. Meta-mordanting: Mordant in combination
be kept in the rack with proper arrangement
with dye is used. and label (Box 7.6). The list of the reagents
3. Post-mordanting: The dye material is applied should be in the laboratory catalogue. The
first followed by mordant. glass bottle is the best container to store the
reagents. The use of amber-coloured bottle is
Example: Haematoxylin itself is a poor dye. preferable for the dye that reacts with light.
However the combination of mordant such as Frequently used reagents can be kept in small
aluminium and haematoxylin makes a stronger glass bottle or Coplin jar. A microscope is nec-
dye. essary to check the stain. Automated strainer
can stain large batches of slides containing
more than 100 slides. Many laboratory prefers
7.6.1 Accentuators manual staining for small batches of slides. In
that case, the glass troughs are used. It is pref-
Accentuators are the group of substances that erable to use the series of sequential arrange-
help to increase the staining intensity of the dye. ment of glass troughs for staining. All the
Accentuators neither form any dye lake nor do troughs should be well covered to prevent
they take part in any chemical reaction. The com- evaporation of the reagents particularly alco-
mon example of accentuator is using potassium holic solution. In addition the laboratory
hydroxide in methylene blue solution. should have ample supply of distilled water.
OH OH
O O
8.2 Haematoxylin HO CH2 HO CH2
C OH C OH
Haematoxylin is extracted from the bark of CH CH2 CH CH2
Haematoxylum campechianum tree that is mainly
seen in the Campeche state of Mexico. Presently Oxidation
known as ripening. The oxidation process is Table 8.1 highlights different types of haema-
slow and takes approximately 3 months. toxylin with their mordant and properties.
However, the useful staining life of the dye is
longer by natural ripening process.
(b) Chemical: This is done by treating the dye 8.3 Bluing
with hydrogen peroxide or sodium iodate or
mercuric oxide. Sodium iodate is the most The most of the regressive staining of haema-
commonly used oxidizing agent. The con- toxylin needs bluing. The removal of excess
version of haematoxylin to haematein is hydrogen ion from the stain is known as bluing.
instant; however the dye has short useful life Here the haemalum which is soluble is converted
span. to insoluble form. Bluing gives crisp blue colour
of the nuclei. In the process of bluing, the pH of
Dye-Mordant Complex Haematein is a weak the solution is raised to 8.5 (alkaline side). The
anion and cannot combine with nucleic acid in tissue section is treated with alkaline reagent,
the nucleus. When a metallic salt (mordant) is and the acidic reagents are neutralized in bluing
combined with haematein, then a cationic dye- process. Bluing is done by the following
metal complex is formed that behaves as a strong methods:
basic dye and combines with nucleic acid
(Fig. 8.1). The type of mordant determines the • Running tap water for several minutes
type of tissue affinity of the dye and the colour of • Treating the section by Scott’s tap water
(pH is 8): 2–3 min
the stain. Commonly aluminium (Al3+), iron
• Ammonium hydroxide (5%): 2–3 min
(Fe3+), molybdenum, tungsten and lead salts are
• Ammonia vapour: few seconds
used as mordant.
Scott's tap water:
Types of Haematoxylin Haematoxylin can be Sodium bicarbonate: 2 g
classified according to its combination with dif- Magnesium sulphate (anhydrous): 10 g
ferent mordants such as: Water: 1 l
Slowly add magnesium sulphate in water so
1. Iron haematoxylin that it dissolves and heat is dissipated.
2. Alum haematoxylin
3. Tungsten haematoxylin Warning The higher pH of the bluing agent
4. Lead haematoxylin makes the bluing more deeper blue colour
5. Molybdenum haematoxylin quickly. However be careful the tissue section in
6. Only haematoxylin (no mordant attached) high pH may be shed out from the slide.
8.3 Bluing 71
Eosin Y for 2 to 3
Step 6 Counterstain : Eosin minutes
Fig. 8.3 Well-stained
tissue by haematoxylin
and eosin stain
(haematoxylin and eosin
stain X 200)
Fig. 8.4 Pale-stained
nuclei (haematoxylin
and eosin stain X 200)
8.6 Iron Haematoxylin 75
Fig. 8.6 Imperfect
deparaffinization of the
tissue section
(haematoxylin and eosin
stain X 200)
Steps: No. 2:
Ferric chloride: 10 g
• Haematoxylin is dissolved completely in Distilled water: 100 ml
absolute alcohol. No. 3:
• Add distilled water. Iodine: 1 g
• Keep the solution for 4–6 weeks for ripening. Potassium iodide: 2 g
Distilled water: 100 ml
Solution 2 No. 4: Working solution
Ferric ammonium sulphate: 5 g Add:
Distilled water: 100 ml No. 1: 40 ml
Step: Dissolve violet crystal of ferric ammo- No. 2: 16 ml
nium sulphate in distilled water. No. 3: 16 ml
Staining Mix those solutions in order.
Steps:
• Dewax the tissue.
• Absolute alcohol. • Dewax.
• 95% ethyl alcohol. • Serial grade of alcohol for hydration.
• Keep section in mordant solution (solution 2) • Stain by freshly prepared working haematox-
for 60 min. ylin solution for 10 min.
• Rinse in distilled water. • Rinse in water.
• Stain by solution 1 (Heidenhain’s haematoxy- • Differentiation: by 2% ferric chloride.
lin 0.5%) for 60 min. • Wash by tap water.
• Rinse in water. • Remove iodine by 95% ethyl alcohol: 5 min.
• Differentiate in 5% alum solution. • Counterstain: 1% eosin for 1–2 min.
• Wash in running water: 5–10 min. • Dehydrate.
• Dehydrate. • Clean by xylene.
• Clean by xylene. • Mount by DPX.
• Mount in DPX.
Table 8.4 Applications of different haematoxylin stains • Low viscosity; otherwise there may be air
for different purposes bubble formation at the time of putting the
Substances Haematoxylin coverslip. This air bubbles are brownish in
Routine stain in histology Harris colour and are known as a cornflake artefact.
sections and cytology smears haematoxylin
Carbohydrates Mayer’s
haematoxylin
Phospholipid Baker’s acid
haematein Box 8.1Clearing Agent
technique • Xylene (dimethyl benzene)
Fibrin, cross striations of skeletal Tungsten
muscle haematoxylin
Connective tissue fibres Verhoeff’s iron Basic properties:
haematoxylin
Amoeba, microfilaria Iron haematoxylin • Colourless.
Nuclear chromatin Gill’s • Refractive index should be the same as
haematoxylin the mounting media and coverslip.
Photomicrography Heidenhain’s iron
• It gives transparent cytoplasm.
haematoxylin
Counterstaining in Ehrlich’s
immunohistochemistry and haematoxylin Warning: toxic
cytochemistry
78 8 Haematoxylin and Eosin Stain of the Tissue Section
Disadvantages:
Box 8.2 Mounting medium
Aim: to give a protective cover over the • Yellow staining after some time.
smear and to make a permanent bond • Takes time to dry.
between the coverslip and the slides • The basic dyes are poorly preserved.
• The same refractive index of the cover- Synthetic Resins DPX is the most widely used
slip and glass slide (1.52–1.54). synthetic resin with a refractive index 1.523. It is
• Colourless. called DPX as it contains:
• It should quickly dry and stick to the slide.
• Resist the growth of microbes. D = distyrene
• No reaction with the stain or tissue. P = plasticizer (tricresyl phosphate)
• Miscible with clearing agent. X = xylene
• A neutral pH to prevent fading of the DPX is colourless and preserves the stains
stain. very well. It dries also very quickly.
• Low viscosity. DPX should be used liberally over the slide,
and the excess DPX should be wiped off from the
Types of mounting medium: coverslip margin.
• Too small amount of mounting medium: Air Liquid coverslip is equally effective.
bubbles may appear. Nowadays many laboratories use automatic cov-
• Too much amount of mounting medium: It erslip machine.
may spread beyond the edges of coverslip, and
the sample may also float.
References
Monosaccharide CH2OH
O O
CH2OH H
H H
H
OH H H H CH2OH
OH
OH OH
H OH OH OH
Glucose Fructose
CH2OH
Oligosaccharides O O
CH2OH H
H H
H
Glucose Fructose
OH H H H
OH O
H OH Sucrose OH OH
Polysaccharide CH2OH CH2OH CH2OH CH2OH
O O O O
H H H H H
H H H
H H H H
OH H OH H OH H OH H
O O O O
H OH H OH H OH H OH
Glycogen
Acid mucin
Neutral mucin
-PAS positive
-Alcian blue
Alcian blue positive
Alcian blue
positive
negative
PAS Neutral mucin
PAS positive
negative Glycogen
Neutral
Acid mucin PAS positive
mucin PAS positive
Diastase
Diastase
resisitant
sensitive
Neutral
mucin Glycogen
Principle (Fig. 9.3)
• Add potassium metabisulphite (2 g).
• The hydroxyl group (OH) of the carbohydrate • Add activated charcoal (2 g).
molecule is oxidized to aldehyde (CHO) • Keep the solution in the dark.
group by periodic acid.
• These aldehyde groups react with Schiff’s Steps
reagent to form a magenta-coloured compound.
1. Deparaffinize.
Components of Solutions 2. Pass through graded lower concentration of
Solution 1: Periodic acid (1%) alcohol and section/smear to bring in
Periodic acid 1 g water.
Distilled water 100 ml 3. Oxidize with periodic acid (1%) for
Solution 2: Schiff’s reagent 5–10 min.
Basic fuchsin 1 g
4. Clean with water.
Distilled water 200 ml
Potassium metabisulphite 2 g
5. Keep in Schiff’s reagent for 20–30 min.
1 N hydrochloric acid (HCl) 20 ml 6. Clean in running tap water for 5 min.
Activated charcoal 2 g 7. Counterstain with haematoxylin.
8. Wash in tap water for blueing.
Preparation 9. Dehydrate in absolute alcohol.
10. Clear in xylene.
• Dissolve basic fuchsin (1 g) in 200 ml of boil- 11. Mount.
ing distilled water.
• Cool the solution. Result
• Add 1 N hydrochloric acid and mix well. Glycogen and glycoprotein: Magenta colour
86 9 Special Stains for the Carbohydrate, Protein, Lipid, Nucleic Acid and Pigments
Materials Positive for PAS Reaction Glycogen, • Myxoma: Mucin-secreting tumours such as
starch, mucin, reticulin, basement membrane, myxomas are positive for Alcian blue stain.
capsule of fungi, etc. • Others: Mucinous materials in myxoedema,
discoid lupus erythematous lesion, etc. are
Testing Schiff’s reagent also demonstrated by Alcian blue stain.
Add drops of Schiff’s reagent to formalin.
Active Schiff’s reagent will quickly change the Basic Principle Alcian blue is a group of water-
colour of formalin to pink. soluble polyvalent basic dye. The dye is made of a
copper-containing phthalocyanine ring with a cop-
9.3.1.2 Alcian Blue per atom in its centre. Th phthalocyanine ring is
Alcian blue stains acid mucin (in acidic pH 2.5), also attached with four isothiouronium groups that
such as sialomucin and sulphomucin. It stains are positively charged (Fig. 9.4). This positively
mucin of the salivary glands, prostate and large charged Alcian blue dye complex has an attraction
intestine. Alcian blue also stains proteoglycans of with anionic sites of the mucin. Copper imparts
cartilaginous material. the blue colour of the dye-mucin complex.
Indications Solution
N
N
N
Mucin
Fig. 9.4 Schematic
diagram shows basic
principle of Alcian blue N
stain. Copper-containing
phthalocyanine ring is
attached with four N
positively charged N N
isothiouronium groups Isothiouronium
that have an attraction group
with anionic sites of the
mucin. Copper imparts
the blue colour of the
Pthalocyanine ring
dye-mucin complex
9.3 Staining of Different Carbohydrates 87
• Mix neutral fast red in hot water. (Fig. 9.5). This is frequently applied in gastroin-
• Filter. testinal biopsy sections.
9.3.2.1 Mucicarmine Stain [5] • Boil the whole solution by keeping the flask in
Indications hot water bath.
• Cool.
• Mucicarmine stain demonstrates acid mucin. • Filter the solution and preserve it in 4°C. This
• It stains mucin of intestinal adenocarcinoma. will be fit for use for 4–6 months.
• The capsule of fungi such as cryptococci is
stained by mucicarmine. Mucicarmine working solution
O
Glycerol
Triglyceride
HO C R-3
Fatty acids
Sphingomyelin Lipofuschin
Fats
Glycolipid
Phospholipid
Oil red O
Ferric
& Sudan black
Haematoxylin
Sudan black
Solution
Steps
Trichloroacetic acid 25 g
• Deparaffinize. Distilled water 100 ml
• Pass through graded alcohol to bring in water.
• Rinse in distilled water: 10–15 dips. Mix it well.
• Dip in a mixture of equal parts of Solution A
Ferric chloride 1 g
and Solution B: 30 min.
Distilled water 10 ml
• Multiple dips in distilled water.
• Counterstain with neutral red for 15 s.
• Rinse in distilled water: 10–15 dips. Mix it well.
• Dehydrate in graded alcohol. Now mix 100 ml aqueous trichloroacetic acid
• Clean in xylene. with 10 ml aqueous ferric chloride solution and
• Mount. keep the mixture in a dark bottle.
Van Gieson stain solution
Result (Fig. 9.10)
Acid fuchsin 100 mg
Hemosiderin: Blue Aqueous saturated picric acid 100 ml
Nuclei: Red
Fig. 9.10 Perls’
reaction in the cytology
smear shows dark blue
haemosiderin pigments
94 9 Special Stains for the Carbohydrate, Protein, Lipid, Nucleic Acid and Pigments
Fig. 9.11 Fouchet’s
stain: The stain
highlights bile cast
within renal tubules in a
case of bile cast
nephropathy (200×)
• Deparaffinize. Steps
• Pass through graded alcohol to bring in water.
• Rinse in distilled water: 10–15 dips. • Deparaffinize.
• Dip the slides in silver nitrate solution (2%) in • Pass through graded alcohol to bring in water.
a Coplin jar: overnight. • Rinse in distilled water for 10–15 dips.
• Drain out the silver nitrate solution. • Dip the slides in ammoniacal silver nitrate in a
• Keep the slides in reducing solution for Coplin jar overnight.
1–2 min. • Wash with distilled water three times.
• Wash in distilled water. • Keep in aqueous sodium thiosulphate (5%):
• Dehydrate in graded alcohol. 2 min.
• Clean in xylene. • Wash thoroughly in running tap water: 2 min.
• Mount. • Counterstain with neutral red (0.5% aqueous):
5 min.
Result • Wash in distilled water.
Argyrophilic cells: Black • Dehydrate in alcohol.
Background: Golden yellow • Clean in xylene.
• Mount.
Fig. 9.12 Schmorl’s
stain: Microphotograph
showing the hyphae of
the dematiaceous fungi
(phaeohyphomycosis);
Schmorl’s stain
highlights the peacock
green-coloured
pigmented fungi (1000×
oil immersion)
Fig. 9.13 Brownish
formalin pigments in the
tissue (haematoxylin and
eosin ×200)
Steps References
• Deparaffinize. 1. McManus JFA. Histological demonstration of mucin
• Pass through graded alcohol to bring in water. after periodic acid. Nature. 1946;158:202.
2. Southgate HW. Notes on preparing mucicarmine. J
• Rinse in distilled water: 10–15 dips. Pathol Bacteriol. 1927;30:729.
• Flood the section with silver nitrate: 1 h in 3. Lillie RD, Ashburn LL. Supersaturated solutions of
strong sunlight. fat stains in dilute isopropanol for demonstration of
• Wash thoroughly in running tap water. acute fatty degeneration not shown by Herxheimer’s
technique. Arch Pathol. 1943;36:432–40.
• Keep in sodium thiosulphate solution: 5 min. 4. Sheehan DC, Hrapchak BB. Theory and practice of
• Wash thoroughly in distilled water. histotechnology. 2nd ed. St. Louis: Mosby; 1980.
• Counterstain with neutral red (0.5%): 5 min. p. 204–5.
• Dehydrate in alcohol. 5. Elleder M, Lojda Z. Studies in lipid histochemis-
try. XI. New, rapid, simple and selective method for
• Clean in xylene. the demonstration of phospholipids. Histochemie.
• Mount. 1973;36(2):149–66.
6. Delamater ED, Mescon H, Barger JD. The chem-
Result istry of the Feulgen reaction and related histo-
and cytochemical methods. J Invest Dermatol.
Calcium deposits: Black
1950;14(2):133–52.
Nuclei: Red 7. Unna PG. Eine Modifikation der Pappenheimschen
Farbung auf Granoplasma. Monatshefte für Praktische
Dermatologie. 1902;35:76.
8. Hall MJ. A staining reaction for bilirubin in sections
9.10.5 Formalin Pigment of tissue. Am J Clin Pathol. 1960;34:313–6.
9. Grimelius L. A silver nitrate stain for alpha-2 cells
Formalin pigment is brownish black in colour. in human pancreatic islets. Acta Soc Med Ups.
Acidic formalin fixation commonly produces for- 1968;73(5–6):243–70.
10. Lillie RD. Histopathologic technique and practical
malin pigment (Fig. 9.13). The pigment can be
histochemistry. 2nd ed. New York: Blakiston; 1954.
removed by alcoholic picric acid. Using buffered 11. von Kossa J. Ueber die im Organismus kunstlich
formalin helps to reduce formalin pigment. erzeugbaren Verkalkungen. Beit Path Anat Allg
Pathol. 1901;29:163.
12. Meloan SN, Puchtler H. Chemical mechanisms
9.10.5.1 Malarial Pigment of staining methods: Von Kossa’s technique: what
Malarial pigment is similar to formalin pigment. von Kossa really wrote and a modified reaction for
This is also brownish black in colour. Unlike for- selective demonstration of inorganic phosphates. J
malin pigment, malarial pigment is intracellular Histotechol. 1985;8:11–3.
13. Corfield AP. Mucins: a biologically relevant glycan
in location. RBCs are usually loaded with malar-
barrier in mucosal protection. Biochim Biophys Acta.
ial pigment. This pigment can be removed by 2015;1850(1):236–52.
treating the section with alcoholic picric acid. 14. Lau SK, Weiss LM, Chu PG. Differential expression
of MUC1, MUC2, and MUC5AC in carcinomas of
various sites: an immunohistochemical study. Am J
9.10.5.2 Starch Clin Pathol. 2004;122(1):61–9.
Starch pigment is produced by talcum powder 15. Gendler SJ, Spicer AP. Epithelial mucin genes. Annu
used in the gloves of the surgeons. The pigment is Rev Physiol. 1995;57:607–34.
positive for PAS stain.
Connective Tissue Stain: Principle
and Procedure 10
Connective tissue is one of the major types of tis- Transient cells (1) Plasma cells, (2) lympho-
sue that connects the different parts of tissue and cytes, (3) neutrophils, (4) monocytes, (5) eosino-
also supports the body parts. Mature connective phils and (6) basophils
tissue is classified as:
than collagen I. Collagen III is present in the 10.1.2 Reticulin Fibres
basement membrane of various organs.
Collagen IV: They are fine fibrillar structures Reticulin fibres are the fine branching fibres that
that are randomly arranged. Collagen IV fibres are interwoven within closely. They are actually
are present in the glomerular basement mem- collagen III fibre. These are argyrophilic fibres.
brane of the kidney. Reticulin fibres support the parenchymal tissue
Collagen V: This is a fibril-forming collagen of the liver, spleen and lymph node.
and is present in minor quantities in specific tis-
sue such as placenta and atherosclerotic plaque.
It is also present in the interstitial tissue of the 10.1.3 Elastic Fibres
kidney and inter-alveolar septum of the lung.
Collagen VI: This is a unique member of col- These are fine fibres. They are present as branch-
lagen family. Collagen VI is connected with vari- ing fibres or sheet. The elastic fibres are made of
ous components of extracellular matrix material. microfibrils that are organized in complex pattern
Table 10.1 highlights the different types of with the help of calcium. Myofibrils of the elastic
collagen. fibres interact with various proteoglycans that
help in the integration of the supporting tissue.
Table 10.1 Comparison of the different types of Elastic fibres provide the elasticity of the blood
collagen vessels, lung and skin.
Types of Fibrillary
collagen pattern Location Function
I Fibrillar, Skin, bone, Gives tensile 10.1.4 Basement Membrane
thicker dentin, tendon strength
II Fibrillar, Cartilage Gives tensile
thin strength Basement membrane is the connective tissue ele-
III Fibrillar, Blood vessels, Provides ments that separate the epithelial and endothelial
thin, lymph node, structural cells from the underlying connective tissue. The
associated lung framework of basement membrane is divided into three layers
with type I the lymph
from cell membrane to away:
collagen node and
spleen and
gives tensile 1. Lamina lucida: This is nearer to the surface
strength to cells and is made of various carbohydrate
various
materials. The lamina lucida contains integrin,
connective
tissues laminin and collagen. This layer may be sim-
IV Network Basement Makes the ply an artefact.
forming membrane framework of 2. Lamina densa: This is the next zone of basement
lamina densa membrane and consists of predominantly colla-
to provide
support gen IV, proteoglycan, laminin and fibronectin.
V Fibrillar Present along Gives tensile 3. Lamina fibroreticularis: This is the fibrous
with type I, strength component and merged with the underlying
placenta, connective tissue elements. They are composed
interstitial
of bunch of microfibrils and collagen fibres.
tissue of the
kidney and
inter-alveolar
septum of the 10.2 Stains
lung
VI Beaded Connective Possibly helps
filaments tissue of the in the 10.2.1 Masson Trichrome [1]
blood vessels, attachment of
uterus, etc. cells and Many different colours of dye are used in Masson
present along connective trichrome stain to differentiate the collagen
with type II tissue
fibres, muscle, fibrin and RBCs.
10.2 Stains 101
Fig. 10.1 Masson
trichrome (MT) stain:
the stain highlighting the
portal tract bluish green,
while the hepatocytes
were stained red. The
nuclei of the cells took a
deeper shade of blue
(normal liver tissue)
(200×)
Van Gieson’s stain solution • Liver biopsy: Reticulin stain helps to demon-
Acid fuchsin 1% (aqueous) 10 ml strate the early cirrhosis. It is an essential stain
Picric acid (aqueous saturated) 100 ml in liver sections.
10.2 Stains 103
Fig. 10.3 Schematic
diagram shows
mechanism of reticulin Silver
stain. Potassium salt
permanganate oxidizes
the carbohydrate
component of the
reticulin fibres to Basic
generate aldehyde Reticulin Reticulin pH
CH2OH CHO
group. In the basic
medium, the silver salt
CH2OH CHO Sodium
produces metallic silver thiosulphate
that reacts with the
aldehyde group of the
Metallic Removes
tissue. Gold chloride Oxidation by Potassium
silver excess
makes this metallic permanganate
unreactive
precipitation permanent. silver
In addition, sodium To make the reaction
thiosulphate is used to permanent
remove the excess
unreactive silver
Gold
chloride
Fig. 10.4 Reticulin
stain: reticulin stain in
the liver highlighting the
normal reticulin pattern
of the liver (400×)
• Deparaffinize.
10.3.1 Verhoeff’s Stain for Collagen [4] • Graded alcohol to bring in water.
• Rinse in distilled water: 10–15 dips.
Principle The haematoxylin dye binds with the • Verhoeff’s iron haematoxylin: 20 min.
elastic tissue by ionic interaction. Ferric salt acts • Differentiation: By 2% ferric chloride. This is
as an oxidizer and helps in binding of haematox- a crucial step. Allow to differentiate till the
ylin and elastic fibres. fibres take black colour. Check the colour by
microscope.
Control: skin tissue • Wash in water.
Solutions • Remove iodine by washing in 95% alcohol.
• Counterstain: Van Gieson’s stain—2 min.
Solution 1 • Rapid dehydration.
Haematoxylin 5 g • Clear in xylene.
Absolute alcohol 100 ml • Mount.
106 10 Connective Tissue Stain: Principle and Procedure
Steps of staining
10.3.2 Weigert’s Resorcin-Fuchsin
Stain [5] • Deparaffinize.
• Graded alcohol to bring in water.
Solution • Wash in water.
Basic fuchsin 2 g • Orcein solution: 30 min (in 56 °C).
Resorcin 4 g • Differentiate: by 1% acid alcohol.
Distilled water 200 ml • Wash in water.
• Counterstain: methylene blue.
• Mix basic fuchsin and resorcin in 200 ml dis- • Rapid dehydration.
tilled water and boil. Now add 25 ml of 30% • Clear in xylene.
ferric chloride in this boiling solution. • Mount.
• Boil for another 5 min
• Cool Result
• Filter Elastic fibres: brown
• Take the precipitate in a filter paper Fibrin and cross striation of the muscle
• Now take 200 ml of 95% ethanol in a flask and Fibrin is best stained by phosphotungstic acid
dissolve the precipitate by heating haematoxylin (PTAH).
• Remove the filter paper
• Add 4 ml of concentrated HCl
Steps of staining 10.3.4 Phosphotungstic Acid
Haematoxylin (PTAH) [6, 7]
• Deparaffinize.
• Graded alcohol to bring in water. Aim PTAH aims to stain fibrin, cross striation of
• Rinse in distilled water: 10–15 dips. the muscle and glial fibres.
• Put the section in the stain solution for 1–3 h
in room temperature. Solutions
• Wash in water.
Haematoxylin 1 g
• Differentiate: by 1% acid alcohol.
Phosphotungstic acid 20 g
• Wash in water. Distilled water 1000 ml
• Counterstain: Eosin.
• Rapid dehydration.
• Clear in xylene. • Dissolve haematoxylin and phosphotungstic
• Mount. acid separately in distilled water by gently
Result heating.
Elastic fibres: purple • Cool.
• Combine the two solutions.
Fig. 10.5
Phosphotungstic acid
haematoxylin (PTAH)
stain: This stain
highlighted the fibrin
thrombi as
haematoxyphilic blue
showing numerous
glomerular fibrin
thrombi (400×)
Solution • Deparaffinize.
1% sodium hydroxide • Pass through graded alcohol to bring in
Sodium hydroxide 1 g water.
Distilled water 100 ml • Rinse in distilled water: 10–15 dips.
Saturated sodium chloride in ethanol (80%) • Congo red: 5 min.
80% ethanol 100 ml • Differentiation by alcoholic potassium
Sodium chloride 1 g hydroxide: Few seconds.
Alkaline alcohol sodium chloride solution
• Wash in water.
Sodium hydroxide (1%) 1 ml
• Counterstain: Alum haematoxylin.
Sodium chloride ethanol (80%) 100 ml
Freshly made solution should be used • Running tap water for blueing.
Alkaline Congo red stock solution • Wash by distilled water.
Congo red 0.5 g • Rapid dehydration.
Alkaline alcohol sodium chloride solution 300 ml • Clear in xylene.
Working solution of Congo red • Mount.
Alkaline Congo red stock solution 100 ml
1% sodium hydroxide 1 ml Result
Use only fresh solution Amyloid: Red colour
References 111
Microbial organisms or microbes are sub- that helps to retain the dye crystal violet on the
microscopic infective organisms that may pro- cell wall. Now Gram’s iodine is added that helps
duce disease in human. to bind the crystal violet on the wall of the bacte-
Broadly these microbes can be subdivided ria. This dye-iodine complex does not diffuse out
into (1) bacteria, (2) fungi, (3) protozoa, (4) hel- in the presence of acetone. Therefore, Gram-
minths and (5) virus. positive organism shows blue colour even after
Routine haematoxylin and eosin stain may not acetone treatment. The dye-iodine complex is
distinctly identify the microbial organism in tis- diffused out easily from the Gram-negative bac-
sue or smear, and therefore special stain is teria and so it becomes colourless. The counter-
needed. Special stain helps to delineate the mor- stain carbol-fuchsin is applied to give
phology and characteristic colour of the organ- Gram-negative organism a pink colour.
isms [1–3] (Table 12.1). It is advisable to have
culture of the microbes if possible. Reagents
Crystal violet solution
Crystal violet 1.0 g
12.1 Bacteria Absolute alcohol 20 ml
Ammonium oxalate (1%) 80 ml
The common stains of the demonstration of bac- Lugol’s iodine
terial organisms are: Iodine crystal 1.0 g
Potassium iodide 2.0 g
Distilled water 300 ml
• Gram’s stain
Basic fuchsin
• Ziehl-Neelsen stain Basic fuchsin 1.0 g
Distilled water 100 ml
Fig. 12.1 Abundant
acid-fast bacilli in a case
of atypical mycobacteria
(Ziehl-Neelsen stain
×1200)
116 12 Stains for the Microbial Organisms
Fig. 12.2 Abundant
lepra bacilli in a lymph
node aspirate in Fite
acid-fast stain for
leprosy (Fite acid-fast
stain for leprosy ×1200)
• Deparaffinize.
This solution can be used for 3 months. • Pass through graded lower concentration of
Methenamine silver working solution alcohol and section/smear to bring in water.
Stock solution of methenamine silver 50 ml • Oxidation: 2% chromic acid, 30 min.
5% sodium borate 5 ml • Wash in distilled water.
12.3 Spirochaetes 117
Fig. 12.3 Methenamine
silver stain for
Pneumocystis carinii
infection (Methenamine
silver stain ×1200)
118 12 Stains for the Microbial Organisms
Result Result
Spirochaetes: Black colour. Background: Viral inclusion: Bright red. Background:
Brown to yellow Yellow
Endocervical canal
Box 13.1: Cervical Smear Collection
Ectocervix
Basic precautions
Collection devices
For LBC: Cervix brush, LBC collection
Fig. 13.3 Schematic diagram of cervical smear collec- fluid in vial
tion. The brush is introduced in the cervical canal the tip
of the brush remains in the endocervical canal and the For conventional:
brooms are touched in the ectocervix. The brush is rotated
and then withdrawn
• Endocervical broom stick, plastic spat-
ula or wooden spatula
–– Rotate the spatula in a complete round turn. • Clean glass slides
–– Withdraw it gently. • Permanent slide marker
–– Spread the sample on the spatula on the • Fixative: 95% ethyl alcohol
glass slide.
–– Immediately fix the slide in 95% ethyl Collection proper
alcohol. Position of the patient: Dorsolithotomy
• For LBC preparation Preparation
–– Use cervix brush.
–– Introduce the central part of the broom into • Open the vagina by speculum for proper
the endocervical canal so that the shorter visualization of the cervix.
bristles of the broom touch to the ectocer- • Clean the vagina by wet swab with
vix (Fig. 13.3). water.
–– Rotate the broom four to five times in a • Insert the cervix brush or plastic spatula
clockwise direction. within in the vagina.
–– Withdraw the broom and put it in the fixa- • Rotate full circle.
tive solution as given by the company. • LBC: Rinse the brush in the fluid within
• Label the glass slide or the vial (for liquid- the vial.
based cytology). • Conventional:
Vaginal smear: The cytology sample is col- –– Spread the material on the glass
lected from the lateral vaginal wall by using a slide.
spatula. –– Immerse the smear immediately in
Endometrial aspiration smear: With the help 95% ethanol for fixation.
of a sterile cannula and a syringe, the endometrial
sample is aspirated.
124 13 Cytology Sample Procurement, Fixation and Processing
13.2.2 Respiratory Samples [3, 4] • The stylet is withdrawn, and aspiration is done
by applying negative suction.
The respiratory samples include sputum, bron- • The needle is withdrawn.
chial brush, bronchial brush, bronchoalveolar • The inside material is ejected by reintroducing
lavage, transbronchial needle aspiration cytology the stylet within the needle.
and fine needle aspiration cytology (FNAC) • Multiple smears are made (both air-dried and
under radiological guidance. alcohol-fixed).
Sputum sample
Gastrointestinal Tract Gastric Brush, Lavage
• Collect the morning sputum in a wide- and Transendoscopic FNAC
mouthed container.
• No fixative is needed. Gastric brush:
Voided urine: Preferable for routine cytology • Ethyl alcohol (95%): It is the most commonly
used fixative. Ethanol causes dehydration of
• Collect second voided urine. the cell and mild shrinkage.
• Collect urine in a clean container. • Methanol (100%): Not cost-effective.
• No fixative. • Denatured alcohol: This is unsuitable for human
• Send the sample in the laboratory immedi- consumption and so less chance of misuse.
ately for processing.
Time of fixation
Bladder wash: • At least 15–30 min.
• If necessary, one can keep the smear in fixa-
• Introduce a catheter or cystoscope.
tive for long duration in a closed bottle or jar.
• Wash the bladder by 50–100 ml normal saline.
• Withdraw the solution.
• Sent the sample immediately to laboratory Box 13.2 Fixation
without any preservative. Ideal fixative
Fig. 13.4 Outline
shows the maximum
Sputum Effusion Urine, Gastric
time duration between
fluid CSF aspirate
the collection of smear
and processing
Maximum Maximum
Maximum
time can be time can be
time can
kept: 24 kept: 24 Immediate
be kept:
hours in hours in
1-2 hours
refrigerator refrigerator
Requisition form
13.4 Processing of Laboratory
Samples Name Age Sex
Unique identification of the patient
Date of collection
Processing of a laboratory sample includes the Site
following steps: Procedure of collection
Clinician’s name and contact information
Tests to be done
• Receiving Clinical history
• Preparing smear Chief complaints
Physical findings
• Staining Radiological features
• Mounting and final submission of the slide Important history: Surgery, chemotherapy,
radiotherapy, exposure of chemicals etc.
13.4.1 Receiving the Sample Fig. 13.5 Requisition form of the cytology specimen
Requisition form: The sample should always be • Smear should be properly fixed and
accompanied with a proper requisition form as labelled.
mentioned in Fig. 13.5. • Paper form should be in a separate bag, and
slide should not be wrapped by requisition
13.4.2 Glass Slides and Liquid form.
Sample
Precautions for liquid samples:
The following precautions are essential regarding
the receiving of glass slides: • Container should be air tight.
• Properly labelled.
• Slide should be received in a shock-resistant • Plastic container is preferable than glass
container. container.
128 13 Cytology Sample Procurement, Fixation and Processing
Centrifuge
• Each sample should have a unique laboratory
bar code number. This is separate from the
Less than 1ml (CSF,
unique identification number. Vitreous fluid etc)
• Stick the bar code number on the container,
smears and forms. Cytocentrifuge
Figure 13.6 shows overview of processing of • Put the fluid sample in clean air tight centri-
different samples. fuged tube.
• Rotate the tube at 1500 rounds per minute
(RPM) for 10 min.
13.5.1 Processing of Sputum • Discard the supernatant liquid.
(Fig. 13.7) • Make multiple smears from the sediments.
• Pour the sputum sample in a Petri dish kept on Cytocentrifuge: Small amount of clean fluid
a black background. such as 0.5–1 ml is processed by cytocentrifuge,
• Carefully examined for any tissue fragments e.g. CSF, ureteric urine, vitreous fluid, etc.
or grey-white substance or bloody material. (Fig. 13.9).
• Pick up the tissue fragments by a clean
forceps. • Rotate the sample 1000 rounds per minute for
• Prepare the smears on the clean glass slide. 5 min.
• A thin layer of smear is formed on the glass
slide.
13.5.2 Processing of Fluid: Urine, • Fix the smears in 95% ethanol.
Body Fluids and Lavage
Centrifuge: The fluid of moderate amount (50– Basic Principle of Centrifuge Rapid circular
100 ml) should be processed by centrifugation, movement of a particle around a central axis
e.g. effusion fluid, turbid urine, etc. (Fig. 13.8). generates a centrifugal force that drives the par-
13.5 Processing 129
Fig. 13.7 Schematic
diagram of processing of
sputum
Petri dish
White paper
Pour the specimen on petri dish
Fluid
Centrifuge
Discard
supernatant
Fig. 13.9 Cytocentrifuge machine to process small quan-
fluid tity of sample
Fig. 13.8 Schematic diagram shows processing of fluid RCF = 1.11 × 10−5 × (round per minute)2 × r
specimen
130 13 Cytology Sample Procurement, Fixation and Processing
Filter paper
pressed on slide
Milipore
lower cup
Negative suction
Slide
Sample
Upper cup
Filter paper
Flask
Centrifuge
Pour sample Keep 4 hours
in 10% formalin for fixation
The most commonly used two routinely available 14.1.1 Dyes Used in Papanicolaou’s
stains in the cytology laboratory are Staining
• Cytoplasmic differentiation: It helps in the 1. Rehydration of the smear: With the help of
assessment of cellular differentiation. subsequent dip in the graded concentration of
• Nuclear details seen. alcohol.
• Transparent stain. 2. Nuclear staining by haematoxylin: Harris hae-
• Demonstrates intracytoplasmic keratin. matoxylin is a good rapid nuclear stain.
Subsequent differentiation is done to remove
Progressive method: As mentioned before, in excess haematoxylin by acid alcohol.
case of progressive stain, the nuclei are optimally 3. Bluing: This is done by treating the smear
stained, and the cytoplasm does not take the dye. with running tap water; alternatively weak
Regressive method: In regressive staining, the alkaline solution can be used.
nuclei are intentionally overstained by haema- 4. Cytoplasmic staining by Orange G (OG): As
toxylin dye. Subsequently the excess stain is OG is alcohol-soluble dye, so the smear is again
removed by acid alcohol. brought into alcohol and stained with OG.
Fig. 14.1 Basic
principles of
Papanicolaou’s stain are Rehydration
Nuclear Bluing: Weak
highlighted in this of the smear:
stain: alkaline
diagram By graded
Hematoxylin solution
alcohol
Cytoplasmic
Cytoplasmic Dehydration
stain: Orange
stain: by EA Clearing
G
Mounting
5. Cytoplasmic staining by EA: The cell cyto- 14.1.3.1 R esult (Figs. 14.2, 14.3
plasm is stained as blue-green colour by EA. and 14.4)
6. Dehydration: It is done by absolute alcohol. Nuclei: Dark blue
7. Clearing: Xylene. Cytoplasm: Blue green
8. Mounting: By DPX mounting medium. Keratin: Orange
Figures 14.2, 14.3 and 14.4 describe the nor-
mal cervical cytology smear, high-grade squa-
14.1.3 Papanicolaou’s Staining Steps mous intraepithelial lesion and squamous cell
carcinoma, respectively, of the routine cervical
1. 70% ethanol: 1 min smear. These are the routine cervical smears of
2. 50% ethanol: 1 min the liquid-based cytology preparation in our lab-
3. Distilled water: 5 dips oratory in Post Graduate Institute of Medical
4. Harris haematoxylin: three and half minutes Education and Research, Chandigarh, India.
5. Distilled water: 5 dips
6. 0.25% aqueous solution of hydrochloric Haematoxylin Solution for Papanicolaou’s
acid: few dips Stain
7. Water: 1 min Harris haematoxylin 5 gm
8. Lithium carbonate: one and half minute Distilled water 1000 ml
9. Water: few dips Alum 100 gm
10. 70% ethanol: 2 min Absolute alcohol: 50 ml
11. 90% ethanol: 2 min Mercuric oxide 2.5 gm
12. Orange G: few dips
13. 95% ethanol: 2 min • Dissolve haematoxylin 5 gm in 50 ml
14. EA modified: 2 min alcohol.
15. Absolute ethyl alcohol: 2 min • Mix 100 gm alum in water and boil it to
16. Absolute ethyl alcohol: 2 min dissolve.
17. Absolute ethyl alcohol and: 2 min • Now mix haematoxylin and alum in water and
18. Xylene: 5 min boil.
19. Xylene: till clear • Take out the flask from heat.
20. Mounting in DPX • Put 2.5 gm mercuric oxide in the solution.
14.1 Papanicolaou’s Stain 135
Fig. 14.2
Papanicolaou’s stain of
the normal cervical
smear (SurePath
preparation of
Papanicolaou’s
stain ×240)
Fig. 14.3
Papanicolaou’s stain of
the cervical smear in a
case of high-grade
squamous intraepithelial
lesion (SurePath
preparation of
Papanicolaou’s
stain ×440)
Fig. 14.4
Papanicolaou’s stain of
the cervical smear in a
case of squamous cell
carcinoma of the cervix
(SurePath preparation of
Papanicolaou’s stain
×440)
Table 14.1 highlights the troubleshooting for FNAC smear. Table 14.2 compares the rela-
areas of the Papanicolaou’s stain. tive advantages and disadvantages of MGG stain
over Papanicolaou’s stain.
Destaining and Restaining of the Smear
Steps
• Dip the smear in xylene until the coverslip
drops. 1 . May Grunwald solution: 5 min
• Keep the smear in acid alcohol for 20 min 2. Running water: 1 min
(80 ml of 95% ethanol and 20 ml of 0.5% 3. Giemsa stain: 15 min
hydrochloric acid). 4. Running water: 1 min
• Wash the smear with running water. 5. Air-drying
• Restain.
Clearing and mounting have been discussed in
Chap. 8.
14.3 May Grunwald Giemsa Stain
Storage of Slides
May Grunwald Giemsa (MGG) is a
Romanowski’s stain and is routinely used in • All the positive slides should be stored for
many laboratories. This stain provides excellent indefinite period.
cytoplasmic detail character (Fig. 14.5). This is a • The negative slide should be stored for a mini-
metachromatic stain. MGG is a convenient stain mum of 5 years.
138 14 Routine Staining in Cytology Laboratory
Fig. 14.5 May
Grunwald Giemsa
stained smear in a case
of pleomorphic
adenoma. Note the
metachromatic staining
of the chondromyxoid
substances as deep
magenta-coloured
material (May Grunwald
Giemsa stain ×440)
Table 14.2 Comparison of Papanicolaou’s stain and May Grunwald Giemsa stain
Features PAP stain MGG stain
Nuclear detail Excellent and very good The chromatin pattern cannot be studied
stain for chromatin stain
Keratin demonstration Orange G stains keratin as Cannot be demonstrated
bright orange colour
Metachromasia Not a metachromatic stain Metachromatic stain
Transparency Transparent stain Not a transparent stain
Background mucin or necrosis Not good Good for demonstration of extracellular
substance
MGG May Grunwald Giemsa, PAP stain Papanicolaou’s stain
Reference
1.
Marshall PN. Papanicolaou staining--a review.
Microsc Acta. 1983;87(3):233–43.
Basic Technique of Fine Needle
Aspiration Cytology 15
Fig. 15.1 Comparison
of exfoliative cytology
and FNAC
Characteristics:
Pattern of cells
Overall appearance
Abundant diagnostic cells
Background
FNAC smear
Characteristics:
Cell morphology
Nuclear details
Scanty diagnostic cells
Coplin jar
• Surgical emphysema in lung FNAC
• Rupture of aneurysmal vessel
• Anaphylaxis in case of hydatid cyst Glass slides
• Suitable fixatives: 95% ethanol for fixation of • Chief complaints with duration
slide. • History of previous FNAC
• Additional • Any bleeding disorder
–– Few capped vials containing 10% formalin
solution for cell blocks. Preparation of the Patient The following
–– Few capped vials containing balanced salt measures may help during preparation of the
solution for flow cytometry. patient:
–– Clean sterile vial for culture (bacterial, fun-
gal, etc.). • Explain the whole procedure in brief.
–– Vials for PCR and other molecular • Take proper consent from the patient particu-
techniques. larly for FNAC of deep-seated lesions and
orbit.
• Talk with the patient and give assurance to
15.3 F
ine Needle Aspiration make him/her relax.
Procedure (Box 15.2) • Clean the area of the site of FNAC with a
spirit swab.
Clinical History The following information are
mandatory before FNAC: Aspiration (Fig. 15.3)
• The cytopathologist should personally per-
• Exact site of swelling form FNAC [4].
• Size of the lesion
a b
c d
Fig. 15.3 FNAC technique is demonstrated on dummy and negative suction is given along with to and fro move-
patient: (a) The area of FNAC is cleaned. (b) The needle ment. (c) The material is expelled on the glass slide. (d)
attached with the syringe is introduced into the swelling The smear is made
142 15 Basic Technique of Fine Needle Aspiration Cytology
• Take the pistol handle with attached plastic • The material comes to the hub of the needle by
syringe and needle. capillary pressure of the atmosphere.
• Immobilize the swelling by two fingers of one • Gently withdraw the needle.
of your free hands. • Fill the syringe with air, and attach the needle
• Gently introduce the needle and move the nee- with the syringe.
dle to and fro in the mass. • Expel the aspirated material on the glass slide.
• Apply negative suction by withdrawing the • Make the smears.
plunger.
• Lastly, release of the plunger to stop negative Advantages The major advantages of FNS are:
suction.
• Withdraw the needle. • To get material without any admixture of
• Apply firm pressure in the site of FNAC to blood.
stop any bleeding. • FNS is helpful in vascular organ-like thyroid.
• Retract the plunger to get enough air within • Small swellings are often difficult to fix and
the syringe. can be done by FNS.
• Reattach the needle. • Easy to manipulate the needle as it is light and
• Eject the material on the slide. without any attachment of syringe.
Smear Preparation
• Push the material on a clean glass slide.
• Material should be a few cm away from the Box 15.2: Fine Needle Aspiration Procedure
end of the slide. • Clinical information:
• Needle should be parallel to the slide and little –– Location
bended. –– Size of the swelling
• Make smear by gently pressing a clean glass –– Duration of the lesion
slide over the lower one to spread the –– Major complaints
material. –– Any history of coagulation disorder
• Make 3/4 smears. • Preparation of the patient:
• Keep both air-dried and alcohol-fixed –– Discuss the technique.
smears. –– Take written consent.
• Aspiration:
–– Clean the area of aspiration by spirit.
15.4 Fine Needle Sampling –– Hold the swelling in between the two
fingers to make it immobilized.
Fine needle sampling (FNS) is done without –– Insert the needle within the mass.
using any syringe. No artificial negative suction –– Make to and fro movement of the
is applied, and the material is aspirated with the needle.
help of negative capillary pressure of the atmo- –– Apply negative suction.
sphere [5]. –– Stop the suction.
–– Withdraw the needle.
Steps –– Detach the needle.
• Clean the area by sprit swab. –– Fill the syringe with air.
• Press the swelling in between the two fingers –– Reattach the needle.
to make it prominent. –– Eject the material on the glass slide.
• Introduce a thin bore needle within the –– Make adequate number of smears.
swelling. –– Apply firm pressure on the site of
• Move the needle to and fro in the same direc- FNAC with the help of cotton swab.
tion and also slowly in different directions.
15.5 FNAC of Deep-Seated Lesions 143
Disadvantages
• Costly
• Time taken procedure
• Good radiation exposure
Steps
• Localize the lesion by endoscopic ultrasound.
• Insert the needle with the stylet through the
endoscope to the mass under EUS
guidance.
• Withdraw the stylet and aspirate with the help
of 20 cm3 syringe by applying negative
suction.
• Do FNAC from multiple sites.
• Finally withdraw the needle, and expel the
material on the slide by reintroducing the sty-
let within the needle. Fig. 15.5 Franzen’s guide is fixed with the left index
finger
Others Magnetic resonance imaging (MRI)
uses radiofrequency energy and is free from any Urinary bladder
radiation hazards. MRI-guided FNAC is still not
popular because of longer time to do, high cost
and complicated procedure. In addition,
mammographic- guided FNAC or core needle
biopsy is also popular nowadays. Core needle
biopsy has the added advantage to detect the in
situ carcinoma of the breast, and therefore it has
almost replaced the FNAC of breast [7].
Franzen’s
needle
15.5.4 Complications of Guided
FNAC Rectum
Prostate
There is no significant complication in the guided Fig. 15.6 Schematic diagram showing transrectal fine
FNAC. However, occasionally, there may be needle aspiration cytology with the help of Franzen’s
needle
bleeding, medical pneumothorax or infection.
• Fix Franzen’s guide in the left index finger
with the help of finger stall.
15.6 Transrectal FNAC
• Now introduce Franzen’s guide gently through
of the Prostate
the rectum to the area of the lesion of
prostate.
Transrectal FNAC of the prostate is easy to do,
• Introduce Franzen’s needle with attached
and it can be done in outdoor basis. It samples
syringe within the guide.
from the different areas of the prostate in a single
• Do FNAC with the help of Franzen’s needle
sitting.
and syringe.
• Take out the needle and syringe.
Steps (Figs. 15.5 and 15.6)
• Expel the aspirated material on the slide.
• Keep the patient in his left lateral position
• Make multiple smears.
with the lower leg extended.
• Palpate the prostate first.
146 15 Basic Technique of Fine Needle Aspiration Cytology
16.1 Introduction
Immunohistochemistry (IHC)/immunocyto-
chemistry (ICC) is the technique to visualize rec-
ognition of antigen present in the tissue with the
help of corresponding antibody. Coons et al. first
time applied immunofluorescence technique on
the frozen section by using fluorescence labelled
antibodies [1]. The antibody conjugated with
enzyme acid phosphatase and horseradish per-
oxidase was used first time by Nakane and Pierce
in 1967 [2]. IHC technique was successfully Fig. 16.1 Antigenic determinant site provokes antibody
introduced in routine formalin-fixed paraffin- formation
embedded (FFPE) section by Taylor and Burns in
1974 [3]. Subsequently the development of antibody. The antigen epitope site and antibody-
monoclonal antibody introduced a new era in the binding site have complementary geometrical
immunohistochemistry [4]. However, it took and chemical features (Fig. 16.1). This is respon-
another 10–15 years to have regular routine use sible for the antigen-antibody reaction. This
of IHC in pathology diagnostic laboratory. antigen-antibody reaction is further visualized by
Presently IHC is an essential technique in every attaching certain label to the primary or second-
pathology laboratory. ary antibody.
The basic principle of immunocytochemistry is Antigen Any substance that is capable of pro-
to demonstrate the specific antigen in the cell by ducing an immunogenic response is called as
applying the corresponding antibody to have antigen. There are specific set of chemical com-
antigen-antibody reaction. The antigen contains ponents that evoke immunogenic response of the
an epitope or antigenic determinant site that antigen which is known as epitope or antigenic
evokes specific immunologic response to develop determinant site.
Antibody (Immunoglobulin) Antibody is also cell. Each hybridoma cell produces only a spe-
known as immunoglobulin. The antibody is pro- cific antibody for a specific antigen.
duced by plasma cells in response to antigenic
stimulation. Immunoglobulin has specific affinity Polyclonal Antibody Polyclonal antibody is
against the epitope of the antigen. Each immuno- generated from the different B lymphocytes in
globulin is composed of a pair of light chains and response to the different epitopes of a single anti-
a pair of heavy chains polypeptides. The light gen. As they are generated from the different
chains of immunoglobulin are of two type κ clone of B cells, these antibodies are known as
(kappa) and λ (lambda). There are five types of polyclonal. There is a chance of batch-to-batch
heavy chain, α (alpha), γ (gamma), δ (delta), ε variation in case of polyclonal antibody.
(epsilon) and μ (mu), and depending on the nature
of heavy chain, immunoglobulins are labelled as The differences between the two types of anti-
IgA, IgG, IgD, IgE and IgM, respectively. The bodies are highlighted in Table 16.1.
antibody is a Y-shaped structure. The two tips of
the Y are known as antigen-binding site of the Affinity Affinity represents the strength of the
immunoglobulin (Fig. 16.2). The base of each binding capacity of the antigenic epitope with the
arm of Y is the hinge region that is flexible region corresponding site of the antibody. It is actually
of the immunoglobulin. the three-dimensional fit of the epitope site of the
antigen with antibody.
Hybridoma Technique In this technique abun-
dant unlimited amount of pure homogenous Avidity Avidity means the overall functional
immunoglobulin is produced. The antibody pro- strength of binding capacity of antibody and anti-
ducing B lymphocyte is fused with a malignant gen complex. The polyclonal antibody reacts
immortal plasma cell. The resultant hybrid cell with multiple epitope sites of the antigen, and
acquires the capability of unlimited proliferation therefore the overall strength of antigen-antibody
and production of specific antibody. The resultant complex is strong. The avidity of an antibody
antibody in this condition is known as “monoclo- depends on these factors:
nal antibody”. They are named as “monoclonal”
because they are produced from a single clone of 1. Valency: The more valency of the antibody,
the greater is the avidity.
2. Affinity: The affinity between the individual
Antigen binding site epitope of the antigen and the corresponding
antigen-binding site of the antibody.
NH2
Table 16.1 The differences between monoclonal and
polyclonal antibody
Light chain
Monoclonal antibody Polyclonal antibody
High production cost Low production cost
Hinge region
Specialized training is No special training is
required to produce required to produce
Heavy chain Directed to a particular Directed to multiple
epitope of an antigen epitopes of an antigen
No variation of batch to May vary from batch to
batch batch
COOH
High specificity Low specificity
Fig. 16.2 Schematic diagram of immunoglobulin mole- Less robust of detection More robust in detection
cule. The antibody is a Y-shaped structure, and the two as the antibody is as the antibodies are
tips of the Y are known as antigen-binding site of the directed to a single directed to multiple
immunoglobulin epitope epitopes
16.2 Detection System 151
16.2.1 Peroxidase-Antiperoxidase
Method (Fig. 16.5)
Primary antibody
labeled Peroxidase-antiperoxidase reagent is an immune
Antigen complex substance. It consists of horseradish per-
oxidase antigen and antibody against horseradish
Direct method peroxidase. Here a secondary bridging antibody is
used between the primary antibody and peroxi-
Fig. 16.3 Schematic diagram of direct immunostaining.
In this method the primary antibody is directly tagged dase-antiperoxidase complex. The primary anti-
with an enzyme or fluorescence body and peroxidase-antiperoxidase reagents are
152 16 Immunocytochemistry in Histology and Cytology
Biotin
Antigen
Secondary
antibody Fig. 16.6 Schematic diagram of avidin-biotin immunos-
taining. In this method the secondary antibody is tagged
Primary antibody with biotin, and avidin conjugated with horseradish per-
oxidase is used. The biotinylated secondary antibody
Antigen tightly binds with the peroxidase-conjugated avidin
Advantage:
Primary antibody
1. High degree of sensitivity: peroxidase-
antiperoxidase method is 1000 times more
sensitive than the indirect conjugated method. Fig. 16.7 Avidin-biotin-conjugated staining. In this
method, a preformed complex of avidin-biotin and horse-
radish peroxidase is used
16.2.2 Avidin and Biotin Method
2. The affinity of the biotin and avidin may vary
The biotin has the high affinity for avidin. In this
widely in different batches. This may signifi-
method the secondary antibody is tagged with
cantly affect the sensitivity and reproducibil-
biotin. Now avidin conjugated with horseradish
ity of the test.
peroxidase is used. The biotinylated secondary
antibody is tightly bound with the peroxidase-
conjugated avidin (Fig. 16.6).
16.2.3 Avidin and Biotin
Conjugated Procedure
Advantage:
• Primary antibody
• Biotinylated secondary antibody
• Preformed complex of avidin-biotin and
Anti-alkaline
horseradish peroxidase phosphatase
Advantage: Alkaline
phosphatase
1. Highly sensitive
Disadvantage:
Secondary
1. Endogenous biotin may cause false-positive antibody
reaction.
Primary antibody
Antigen
16.2.4 Biotin-Streptavidin Method
Fig. 16.8 Alkaline phosphatase-antialkaline phosphatase
method. In this method, a complex of alkaline phosphatase-
In this system the avidin is replaced by the tet- antialkaline phosphatase (APAAP) is used. The secondary
rameric antibody streptavidin that is directly antibody or linking antibody is used to bridge the primary
conjugated with enzyme. The streptavidin mol- antibody and APAAP complex
ecule has very high affinity against biotin. The
biotin-
streptavidin complexes give better antibody in the APAAP complex are both from
amplification and detection than avidin-biotin the same species, whereas the secondary linking
complex. antibody is from a different species.
Advantages: Advantages:
• Streptavadin does not cross react with the 1. It is used in cases where the tissue contains high
lectin-like substances. quantity of endogenous peroxidase such as in
• The enzyme is more stable and can be stored the bone marrow, lymph node, etc. [5]. It is often
for longer duration as this is directly bound used in dual immunostaining such as APAAP
with streptavidin. and peroxidase-antiperoxidase staining.
• There is no non-specific electrostatic binding 2. APAAP method provides distinct bright red
with streptavidin as its isoelectric point is near colour which is easy to identify compared to
to neutrality. conventional peroxidase stain.
3. APAAP is stable for long duration.
Steps:
Dextran
polymer • Do initial horseradish peroxidase-conjugated
system.
• Add biotinylated tyramine solution.
Secondary • Wash.
antibody
• Add horseradish peroxidase-conjugated
streptavidin.
Primary antibody
• Add chromogen (DAB) to visualize the
Antigen reaction.
Fig. 16.9 Polymer-based labelling method. In this tech- The comparison of different visualization
nique a polymer backbone (dextran) is used, and a large techniques in immunohistochemistry is high-
number of enzyme molecules and secondary antibodies
are conjugated within the polymer. The primary antibody lighted in Table 16.2.
followed by dextran-enzyme secondary antibody complex
is applied to enhance the sensitivity
16.3 T
he Sample of Tissues
primary antibody is applied followed by dextran- for Immunocytochemistry
enzyme secondary antibody complex [7].
16.3.1 Histopathology
Advantages:
• Formalin-fixed paraffin-embedded tissue
1 . This is simple and rapid two-step technique. • Frozen section
2. Compared to avidin-biotin technique, this is
more sensitive because the large number of
enzymes takes part in a single binding of pri- 16.3.2 Cytology
mary and secondary antibody.
3. No background staining as it bypasses biotin • Cell block tissue
and avidin bindings. • Smear from liquid-based cytology
• Direct cytology smear: imprint or FNAC
smear
16.2.7 Catalysed Signal • Cytospin smear
Amplification (Tyramine
Signal Amplification)
16.3.3 Sample Collection
Catalysed reporter deposition technique is also
known as tyramine signal amplification (TSA) Histopathology
method [8]. This technique exploits the catalytic Formalin-fixed paraffin-embedded (FFPE) tissue:
property of HRP in the presence of hydrogen perox- This is the most popular sample for IHC. Presently
ide. Biotinylated tyramine is used in the presence of most of the antibodies work on FFPE tissue section.
16.3 The Sample of Tissues for Immunocytochemistry 155
T
deposited only in the T
T T
T
antigen-antibody
T
reaction site. This biotin HRP
T
is visualized by T
T
avidin-biotin technique
Secondary antibody
Primary antibody
Antigen
Table 16.2 (continued)
Name of Primary Secondary
technique antibody antibody Visualization system Advantages Comments
Polymer-based Primary Ab Secondary Ab Polymer backbone • Simple and
labelling method conjugating with rapid two steps
large number of technique
enzyme molecules • More sensitive
and more than 20 because the
secondary Abs large number of
enzymes takes
part in a single
binding of
primary and
secondary Ab
• No background
stain
Tyramine signal Primary Ab Secondary Ab Biotinylated • Good sensitivity • Time consuming
amplification tagged with tyramine reacts in • Non-specific
HRP the presence of HRP background stain
and hydrogen may appear
peroxide to generate
activated biotinylated
tyramide that reacts
with tissue tyrmine
Ab antibody, HRP horseradish peroxidase
Frozen section: The use of frozen section for Direct Smear The smears from the FNAC or
IHC is now discouraged. other sample are made directly and fixed immedi-
ately. The direct smear gives poor quality of the
Cell Block The cell block from the cytology immunostaining as there may be severe back-
material is preferable in case of cytology sample. ground artefact after immunostaining.
Any specimen can be used for cell block except
the specimen with very low cellularity. Advantages:
Disadvantages:
16.3.5 Antigen Retrieval
• Specimen not fit for bloody and mucoid
materials Formalin-fixed tissue often gives inconsistent
• Difficult on scanty cellularity results of IHC. In real life it is very difficult to
• Best choice for lymphoid neoplasm replace formalin as a tissue fixative. Enzymatic
digestion of the tissue also does not always pro-
vide good result. Therefore there is a great need
16.3.4 Fixation to have antigen retrieval in FFPE tissue. The term
“antigen retrieval” (AR) means the recovery of
Appropriate fixation of the tissue/cell is manda- the antigenicity of the tissue which is unmasked
tory to get good IHC. at the time of formalin fixation [10]. Fraenkel-
Conrat et al. showed that the reaction between
Buffered Neutral Formalin Buffered neutral formalin and tissue is to some extent reversible if
formalin (10%) is still now the best fixative for heat is applied [11]. Based on this theory, subse-
tissue and cytology sample. quently different studies have shown that the AR
is possible by heating the tissue [12, 13].
Advantages: The important factors that control the AR:
• Cell morphology is well preserved 1. Heating temperature and time period: There
• Cheap is an inverse correlation between the heating
• Sterilizes the specimen from bacteria temperature and time needed for AR. A test
• Prevents diffusion of protein from the cell by battery approach may settle the optimum tem-
cross-linking the protein perature and time period for successful
AR. However, low temperature (around
Disadvantages: 90 °C) with prolonged heating period helps in
the perseveration of tissue morphology.
1. Buffered formalin is not good for RNA-based 2. pH of the AR solution: Majority of the anti-
IHC. For RNA stain PAX gene, RCL2 or bodies work well if pH is used around 7.4.
Z7-fixative is suggested [7]. PAX gene fixa-
tive gives excellent staining quality for RNA-
based IHC [9]. 16.3.6 Microwave Retrieval
2. Cross-linking of proteins by formalin hinders
the access of the antibody to the epitope of the This is the most effective and popular method of
antigen. Therefore the antigen retrieval is AR [14]. It is always better to standardize the
often necessary for IHC on formalin-fixed tis- technique to get the consistent result. The follow-
sue [10]. ing factors may need attention:
Others Commercially available fixatives are 1. Wattage: The range of wattage of the micro-
used in LBC preparations and they give equally wave oven can be 750–800 W.
good results. Various other fixatives are ethanol, 2. AR buffer solution: Usually 0.01 M citrate
methanol and acetone which are used in cytol- buffer of pH 6 gives good result.
158 16 Immunocytochemistry in Histology and Cytology
In any IHC staining, it is extremely essential to The basic steps of immunocytochemistry are
have proper control because it validates the (Fig. 16.11):
Antigen
Blocking endogenous
Blocking nonspecific Incubation with
enzyme
binding primary antibody
Secondary
antibody Chromogen
Table 16.4 (continued)
Section or smear is dried during • Take proper Box 16.2: Major Applications of
staining precaution to avoid Immunocytochemistry in Histology and
drying of the tissue Cytology
Chromogen is incompletely • Dissolve the • Diagnosis
dissolved chromogen
properly –– Classification malignant round cell
Positive control and test samples tumors
are showing background –– Differentiating carcinoma, sarcoma,
staining, but negative control is melanoma, etc.
free from background stain
–– Diagnosis of malignancy in effusion
Primary antibody is over • Dilute the primary
concentrated antibody properly sample
Only test sample showing –– Sub-classification
background stain Lung carcinomas
Over fixation and possibly the • Standardize the Lymphoma
test sample and controls are proper fixation of –– Diagnosis of soft tissue neoplasm
differently fixed the test sample
Small round cell sarcomas
Spindle cell tumors
16.6 Applications Pleomorphic sarcomas
• Prognosis
IHC is now an essential component in laboratory. –– Cell proliferation analysis
It has widespread use in diagnosis, sub- –– Hormone receptors
classification and prognosis of the tumors. IHC –– Prognostic markers
also helps in the identification of various infec- • Therapeutic applications: Receptor
tive organism of our body. studies for specific therapy
Box 16.2 highlights the major applications of • Infective organisms to identify
IHC.
HBME-1:
16.7 Diagnostic
Immunocytochemistry • HBME-1 stains intensely around the periph-
ery of the mesothelial cells.
16.7.1 Mesothelial Cells Versus • The specificity of HBME-1 is about 80%, and
Adenocarcinoma the majority of the mesothelial cells show
strong positivity.
IHC is often proved as very helpful to differenti-
ate reactive mesothelial cells from adenocarci- Wilms’ tumor gene 1 (WT-1):
noma [15]. Few important mesothelial markers
are briefly outlined here: • This is a transcription factor isolated in the
cells of the kidney.
• It is expressed in the cytoplasm and nucleus of
16.7.2 Mesothelial Markers the mesothelial cells.
• The WT-1 positivity is also seen in desmo-
Calretinin: plastic small round cell tumors and ovarian
serous carcinoma.
• Present in the mesothelial cells, steroid pro-
ducing cells of ovary and testis, renal tubular D2-40:
cells and lipocytes.
• The sensitivity and specificity of calretinin are • It is a relatively sensitive (85%) and specific
about 100% and 80%, respectively [16]. (95%) marker of mesothelial cells.
16.9 Mesenchymal Markers 163
Cytokeratin: Desmin:
• Depending on their molecular weights, cyto- • Desmin is present in smooth muscle, skeletal
keratin is classified into 20 different types. muscle and cardiac muscle.
164 16 Immunocytochemistry in Histology and Cytology
a b c
d e f
Fig. 16.12 Antibody panel helps to determine the exact enlargement and pleomorphism. The immunocytochemis-
sit of origin of the metastatic tumor. A 55-year-old female try panel on cell block sections shows CK 7 (c), PAX 8 (e)
presented with massive ascites. Cytology smear (a) shows and WT 1 (f) positivity and CK 20 negativity (d). The
abundant tight ball like cluster, and cell block section immunocytochemistry result indicates the ovarian origin
shows (b) sheets of malignant cells with moderate nuclear of the carcinoma
• HCG is normally secreted by the syncytiotro- • Thyroid transcription factor-1 (TTF-1) is pres-
phoblast cell of the placenta. ent in follicular epithelial cells of the thyroid,
• The beta subunit of HCG is hormone bronchial epithelial cells of the lung and ade-
specific. nocarcinomas developed from those cells.
a b c
d e f
Fig. 16.14 Antibody panel to determine the type of a 45 (b) and CD 99 (c) and positive for NSE (d), chromo-
malignant round cell tumor. A 13-year-old male presented granin (e) and synaptophysin (f). The result of the immu-
with 5 cm diameter right upper abdominal mass near the nocytochemistry panel indicates the diagnosis of
adrenal gland. The cytology smear (a) shows discrete neuroblastoma
monomorphic round cells. The cells are negative for CD
16.16 Immunocytochemistry for Therapy and Management 167
Table 16.7 Basic panel to differentiate the different malignant round cell tumors
Tumor type CD 45 CD99 Myogenin Pancytokeratin Desmin Chromogranin
NB − + − − − +
EWS/PNET − + − − − +
NHL + − − − − −
RMS − − + − + −
DSRCT − − − + + −
WT − − − + + −
NB neuroblastoma, EWS Ewing’s sarcoma, PNET peripheral neuroectodermal tumor, NHL non-Hodgkin lymphoma,
RMS rhabdomyosarcoma, DSRCT desmoplastic small round cell tumor, WT Wilms’ tumor
a b
c d
Fig. 16.15 The cell block of a case of infiltrating duct carcinoma of the breast (a). Oestrogen receptor (b), progester-
one receptor (c) and Her-2/neu immunostain (d) were done that showed strong positivity
a b
c d
Fig. 16.16 A case of lung carcinoma (a) shows p63 (b) and CK 5/CK6 positivity (c) and TTF-1 negativity (d). This
indicates the diagnosis of squamous cell carcinoma
drugs. Table 16.8 shows the basic panel for the samples: comparison of five molecular fixatives. J
differentiating adenocarcinoma from squamous Clin Pathol. 2013;66(9):807–10.
10. Shi SR, Cote RJ, Taylor CR. Antigen retrieval tech-
cell carcinoma of the lung. niques: current perspectives. J Histochem Cytochem.
2001;49(8):931–7.
11. Fraenkel-Conrat H, Brandon BA, Olcott HS. The
reaction of formaldehyde with proteins. IV: participa-
References tion of indole groups. J Biol Chem. 1947;168:99.
12. Boon ME, Kok LP, Suurmeijer AJH. The MIB-1
1. Coons AH, Creech HJ, Jones RN. Immunological method for fine-tuning diagnoses in cervical cytology.
properties of an antibody containing a fluorescent In: Shi S-R, Gu J, Taylor CR, editors. Antigen retrieval
group. Exp Biol Med. 1941;47(2):200. techniques: immunohistochemistry and molecular
2. Nakane PK, Pierce GB. Enzyme-labeled antibodies morphology. Natick: Eaton; 2000. p. 57–70.
for the light and electron microscopic localization of 13. Stirling JW. Antigen retrieval and unmasking for
tissue antigens. J Cell Biol. 1967;33:307–18. immunoelectron microscopy. In: Shi S-R, Gu J,
3. Taylor CR, Burns J. The demonstration of plasma Taylor CR, editors. Antigen retrieval techniques:
cells and other immunoglobulin-containing cells immunohistochemistry and molecular morphology.
in formalin- fixed, paraffin-embedded tissues Natick: Eaton; 2000. p. 93–113.
using peroxidase-labelled antibody. J Clin Pathol. 14. Shi S-R, Key ME, Kalra KL. Antigen retrieval
1974;27:14–20. in formalin- fixed, paraffin-embedded tissues: an
4. McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre enhancement method for immunohistochemical stain-
JW, Milstein C. A human thymocyte antigen defined ing based on microwave oven heating of tissue sec-
by a hybrid myeloma monoclonal antibody. Eur J tions. J Histochem Cytochem. 1991;39:741.
Immunol. 1979;9:205–10. 15. Delahaye M, van der Ham F, van der Kwast
5. Erber WN, McLachlan J. Use of APAAP technique on TH. Complementary value of five carcinoma markers
paraffin wax embedded bone marrow trephines. J Clin for the diagnosis of malignant mesothelioma, adeno-
Pathol. 1989;42:1201. carcinoma metastasis, and reactive mesothelium in
6. Cordell JL, Falini B, Erber WN, et al. serous effusions. Diagn Cytopathol. 1997;17:115–20.
Immunoenzymatic label of monoclonal antibodies 16. Yaziji H, Battifora H, Barry TS, et al. Evaluation of
using immune complexes of alkaline phosphatase and 12 antibodies for distinguishing epithelioid meso-
monoclonal anti-alkaline phosphatase (APAAP) com- thelioma from adenocarcinoma: identification of a
plexes. J Histochem Cytochem. 1984;32:219. three-antibody immunohistochemical panel with
7. Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli maximal sensitivity and specificity. Mod Pathol.
M, Ceccarelli C, Pieri F, Fraternali-Orcioni G, Pileri 2006;19:514–23.
SA. The EnVision++ system: a new immunohis- 17. Chu PG, Weiss LM. Expression of cytokeratin 5/6 in
tochemical method for diagnostics and research. epithelial neoplasms: an immunohistochemical study
Critical comparison with the APAAP, ChemMate, of 509 cases. Mod Pathol. 2002;15:6–10.
CSA, LABC, and SABC techniques. J Clin Pathol. 18.
Busam KJ, Iversen K, Coplan KA, Old LJ,
1998;51(7):506–11. Stockert E, Chen YT, McGregor D, Jungbluth
8. Toda Y, Kono K, Abiru H, Kokuryo K, Endo M, A. Immunoreactivity for A103, and antibody to
Yaegashi H, Fukumoto M. Application of tyra- melan-A (Mart-1), in adrenocortical and other steroid
mide signal amplification system to immunohisto- tumors. Am J Surg Pathol. 1998;22:57–63.
chemistry: a potent method to localize antigens that 19.
Bodey B, Bodey B Jr, Kaiser HE.
are not detectable by ordinary method. Pathol Int. Immunocytochemical detection of prostate specific
1999;49:479–83. antigen expression in human breast carcinoma cells.
9. Staff S, Kujala P, Karhu R, Rökman A, Ilvesaro J, Anticancer Res. 1997;17:2577–81.
Kares S, Isola J. Preservation of nucleic acids and 20. Saleem TB, Ahmed I. Gastrointestinal stromal tumour-
tissue morphology in paraffin-embedded clinical -evolving concepts. Surgeon. 2009;7(1):36–41.
Flow Cytometry: Basic Principles,
Procedure and Applications 17
in Pathology
Photomultipler Computer
tube
Table 17.1 Fluorochrome dye used in DNA Table 17.2 Fluorochrome dye for conjugating with
antibody
Excitation Emission
maximum maximum Excitation Emission
Flurochrome (nm) (nm) maximum maximum
Propidium iodide 305, 540 620 Fluorochrome (nm) (nm)
Ehidium bromide 493 620 Fluorescein isothiocyanate 495 519
Hoechst 33342 350 461 (FITC) 488
Hoechst 33258 352 461 Phycoerythrin (PE) 496 576
Diamidinophenylindole 359 461 Allophycocyanin (APC) 650 660
(DAPI) Rhodamine Red-X 570 590
Acridine orange 503 530 (DNA), Texas Red® 595 613
640 (RNA) PE-Cy7® 488 566 778
Peridinin chlorophyll 477 678
(PerCP)
nm nano micrometre, 10−9 m
The various types of cytology specimens for 1. Cytology samples are easy to process and
FCM include: require less effort for disaggregation and
thereby single cell preparation is easier to
1. Fine needle aspiration cytology materials:
make for FCM.
Lymph node, breast, lung, prostate, etc. 2. It is relatively easy to procure cytology
2. Exfoliative samples: Effusion fluid, CSF,
sample.
bladder wash
174 17 Flow Cytometry: Basic Principles, Procedure and Applications in Pathology
17.4.5 Flow Cytometric • Wash the cells three times in PBS solution by
Immunophenotyping (FCI) 3000 rounds per minute for 3–5 min.
• Discard supernatant.
Direct stain: • Resuspend the cells in 500 μl PBS solution.
• Run in FCM.
• Centrifuge the sample: 3000 rounds per min-
ute for 3–5 min. Indirect staining procedure [2, 3] (Fig. 17.3):
• Discard the supernatant fluid.
• Re-suspend in citrate buffer. • Collect sample in citrate buffer.
• Prepare single cell suspension by repeated • Wash the cells in PBS (3000 rounds per min-
syringing through nylon mesh or by trypsin- ute for 3–5 min).
ization [1]. • Discard the supernatant and resuspend the
• Keep the cell concentration in the buffer is cells in ice-cold PBS, 3% BSA, 1% sodium
kept as at least 2 × 106 cells per ml. azide.
• Take in 100 μl in PBS (pH 7.4). • Keep 100 μl solution of cells (1.5 × 106 cells/
• Add 10 μl of labelled antibodies for 25 min in ml concentration of cells) into multiple small
the dark place at room temperature. aliquots.
Fig. 17.3 Schematic
diagram explains the
steps of indirect
immunostaining
176 17 Flow Cytometry: Basic Principles, Procedure and Applications in Pathology
• Take 100 μl solution of cells and incubate with –– Cell ploidy analysis: DNA ploidy patter,
50 μl of primary nonconjugated antibody for percentage of S-phase cells
30 min at room temperature in the dark. –– Mention summary of the different findings,
• Wash the cells in PBS. conclusions and further suggestions
• Add 50 μl of a conjugated secondary
antibody.
• Keep for 30 min at room temperature. 17.5 Targets of Application
• Wash three times in PBS.
• Discard the supernatant and resuspend the FCM can be used for quantitative measurement
cells in 500 μl PBS. of various cellular characteristics (Box 17.4).
• Run in FCM. Many of the targets of application of FCM are
research oriented.
The most common applications of FCM in
17.4.6 Data Acquisition [4] clinical laboratory include [5, 6] (Fig. 17.4):
17.6 D
NA Content and Ploidy The different types of aneuploidy are men-
Analysis tioned below:
DNA Ploidy The clone of cells containing the Fig. 17.5 Schematic diagram of different types of aneu-
abnormal amount of DNA is known as aneuploid ploidy in DNA histogram
178 17 Flow Cytometry: Basic Principles, Procedure and Applications in Pathology
peak from normal G2M peak. However, the Bladder Washings DNA ploidy estimation in
presence of more than 20% cell population in the gated population of cytokeratin positive cell
this peak suggests a tetraploid aneuploidy is helpful in the detection of malignancy in fol-
(DNA index is 2). low-up cases of urothelial carcinoma of the blad-
der [8].
Hypertetraploid Aneuploidy (Fig. 17.5) Here
the aneuploid population of cells forms a peak
beyond the G2M peak as they have more than 4n CSF Due to low cellularity, DNA FCM has less
amount of DNA peak (DNA index is greater diagnostic capability in the detection of malig-
than 2). nancy in CSF. It has been demonstrated that the
combined use immunophenotyping and DNA
S Phase The S-phase fraction of cells has 2n–4n FCM is more helpful in the diagnosis of lym-
amount of DNA so they remain in between G0G1 phoma in CSF [9, 10].
and G2M peak. The number of such cells can be
calculated from the DNA histogram.
17.6.3 Prognosis of the Patients
17.6.1 Clinical Application DNA aneuploidy and high S phase are poor prog-
nostic factors in various solid tumors such as
DNA FCM provides usual information on the bladder, prostate, ovarian and endometrial carci-
DNA ploidy and S-phase fraction of the tumor nomas [11].
cell population. The presence of aneuploidy cell
population and/or high S phase suggests a malig-
nant lesion [1, 2]. However, we should keep in 17.7 Immunophenotyping
mind that malignant tumors may often show dip-
loid cell population, and also uncommonly A large number of CDs have been described in
benign tumor may have aneuploidy population of different cells of lymphoid origin [12]
cells. (Table 17.3). Monoclonal antibodies against var-
ious CDs are now also available for the use of
flow cytometric immunophenotyping (FCI)
17.6.2 Diagnosis (Figs. 17.6, 17.7, 17.8 and 17.9). The antibodies
105
105
CD20 PE-A
104
104
PE-A
Q1 Q2 Q1 Q2
103
103
102
102
Q3 Q4 Q3 Q4
Fig. 17.6 Dot plot of CD 45 stain in flow cytometry Fig. 17.8 Dot plot of CD 20 stain (B cell marker) in flow
cytometry
105
105
104
104
CD3 PE-A
PE-A
Q1 Q2
Q1-1 Q2-1
103
103
102
102
Q3-1 Q4-1
Q3 Q4
Fig. 17.7 Dot plot of CD 19 stain (B cell marker) in flow Fig. 17.9 Dot plot of CD 2 and CD 3 stain (T cell marker)
cytometry in flow cytometry
Fig. 17.10 The
diagram highlights the
differences between flow
cytometric
immunostaining versus
immunocytochemistry
17.7 Immunophenotyping 181
Table 17.4 highlights the different CD markers of 1. Cells are washed in 1500 RPM for 5 min in
lymphoma cases. PBS.
2. Resuspend the cells in PBS and keep the
concentration as 1.5 × 106 cells/ml.
3. Take the cells in 60 μL of PBS.
17.7.3 Apoptosis [14, 15] 4. Add 1 ml ice-cold 70% ethyl alcohol drop by
drop.
FCM is one of the important technologies to
5. Keep for 1–2 h in −20 °C for permeabilization.
detect apoptotic cell death. Quantitation of the
6. Wash the cells in PBS by centrifuging.
apoptotic cells is possible with the help of light
7. Discard the supernatant fluid.
scatter (a loss in forward light scatter), plasma
8. Add 1 ml solution containing propidium
membrane changes, and DNA content (a sub-
iodide, RNAse and Triton X. [RNAse 2 mg,
diploid peak).
Triton × 10 ml and PI 0.40 ml of (500 μg/ml)]
9. Yu GH, Vergara N, Moore EM, King RL. Use of 15. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W,
flow cytometry in the diagnosis of lymphoprolifera- Hotz MA, Lassota P, Traganos F. Features of apop-
tive disorders in fluid specimens. Diagn Cytopathol. totic cells measured by flow cytometry. Cytometry.
2014;42(8):664–70. 1992;13(8):795–808.
10. Bromberg JE, Breems DA, Kraan J, Bikker G, van 16. Riley RS, Ben-Ezra JM, Tidwell A, Romagnoli
der Holt B, Smitt PS, van den Bent MJ, van’t Veer M, G. Reticulocyte analysis by flow cytometry and
Gratama JW. CSF flow cytometry greatly improves other techniques. Hematol Oncol Clin North Am.
diagnostic accuracy in CNS hematologic malignan- 2002;16(2):373–420.
cies. Neurology. 2007;68(20):1674–9. 17. Miyoshi H, Arakawa F, Sato K, Kimura Y, Kiyasu
11. Coon JS, Landay AL, Weinstein RS. Advances in flow J, Takeuchi M, Yoshida M, Ichikawa A, Ishibashi
cytometry for diagnostic pathology. Lab Investig. Y, Nakamura Y, Nakashima S, Niino D, Sugita Y,
1987;57:453–79. Ohshima K. Comparison of CD20 expression in
12. Zola H, Swart B, Nicholson I, et al. CD molecules B-cell lymphoma between newly diagnosed, untreated
2005: human cell differentiation molecules. Blood. cases and those after rituximab treatment. Cancer Sci.
2005;106:3123–6. 2012;103(8):1567–73.
13. Dey P. The role of ancillary techniques to diag-
18. Chatterjee T, Mallhi RS, Venkatesan S. Minimal
nose and sub-classifiy non Hodgkin lymphomas residual disease detection using flow cytometry:
on fine needle aspiration cytology. Cytopathology. applications in acute leukemia. Med J Armed Forces
2006;17(5):275–87. India. 2016;72(2):152–6.
14. Darzynkiewicz Z, Bedner E, Smolewski P. Flow
cytometry in analysis of cell cycle and apoptosis.
Semin Hematol. 2001;38(2):179–93.
Digital Image Analysis and Virtual
Microscopy in Pathology 18
Fig. 18.1 Illustrated
figure of the basic steps
of image analysis: (a) at
first the coloured image
is captured. (b)
Subsequently the
coloured image is
converted into grey
image. (c) The cells of
interest are detected by
adjusting the grey value.
(d) Finally the
measurement is done
a b
Coloured image is captured Coloured image converted into grey
image
c
d
Cells of interest are detected
By adjusting grey value Measurement is done
between the objects of interest and the back- • Similarity of the region of image: The
ground. We use adaptive filters or inverse image is segmented according to the sim-
anisotropic diffusions technique for image ilarity and dissimilarity of the data. At
enhancement. first scale-space filtering technique is
( b) Automated segmentation: Automated seg- used followed by watershed clustering
mentation is the most important step to have technique [14].
a successful analysis of the digital images. In –– Scale-space filtering: In this technique
fact, this is the most challenging area of the the filtering is done in a continuum of
digital image analysis. Automated segmenta- scales by applying Gaussian filters.
tion can be done by: –– Watershed clustering technique: This
• Grey level threshold method: As the nuclei is also known as region-based seg-
are differently stained, so they can be sep- mentation approach. The watershed
arated by adjusting the grey threshold lines are constructed around the
[12]. At first, the colour of the different boundary of the different objects of
components of the image is noted such as interest, and this watershed technique
nuclei, cytoplasm and stromal area. Then is applied to determine the individual
the image of the interest is segmented by objects in the image. The major prob-
utilizing the colour and gradient informa- lem of watershed technique is over-
tion [13]. segmentation and thick watershed.
188 18 Digital Image Analysis and Virtual Microscopy in Pathology
Fig. 18.2 Object-based
features of digital image Object based
are highlighted here. features
There are four types of
object-based features:
Texture
(1) geometric features, Geometrical features Co occurrence of Matrix
(2) textural features, (3)
Energy
densitometric features Area, diameter, perimeter
Uniformity
and (4) topological Convex area, convex deficiency,
features Entropy
Major and minor axis length Smoothness
Equivalent diameter, sphericity, Markovian
compactness
Texture
Extent, aspect ratio Run length texture
Wavelets
Densitometric
Optical density, Topological
Integrated optical Voronoi diagram
density, Mean optical Delaunay triangulation
density Minimum spanning tree
Skeleton
(c) Feature extraction and unnecessary data The non-linear data reduction techniques
reduction: The different sorts of features are are:
extracted from the digital images such as
(Fig. 18.2): • Spectral clustering
1. Geometric features: Shape and size • Isometric mapping
2. Topological features: Voronoi diagrams, • Locally linear embedding
Delaunay triangulation and minimum • Laplacian eigenmaps
spanning trees
3. Densitometric study: Optical density, After significant data reduction, the remaining
integrated optical density and mean opti- features can be used as input features to deter-
cal density mine the disease identification, classification or
4. Textural features: Co-occurrence of grading.
matrix (energy, uniformity, entropy,
smoothness), Markovian texture, run Instruments Requirements Nowadays no spe-
length texture, wavelets and fractal cial equipments are needed for image analysis. A
dimension digital camera attached to the microscope and a
It is essential to reduce the vast quantity of suitable software is sufficient for image analysis.
data for the feasible interpretation. Either linear ImageJ software is now freely available in the
or non-linear data reduction techniques can be Internet (https://imagej.nih.gov/ij/download.
used. The linear techniques of data reduction html), and various geometric measurements are
include: possible by this software. There are many other
free softwares available in the Internet to do
• Principal component analysis image analysis.
• Multidimensional scaling
• Linear discriminant analysis
18.4 Morphologic Features 189
18.4 Morphologic Features is available along with fluorescent images data. The
spatial arrangement of the objects is the important
The various morphometric features can be step of the pattern recognition.
detected with the help of digital image analysis
such as (1) geometrical shape and size, (2) densi- The following features are used for the spatial
tometric features and texture, (3) pattern of distri- arrangement of histopathology image analysis:
bution and (4) chromatin specific.
1. The local information around a single cell
Geometrical Shape and Size These features cluster
include cell diameter, perimeter, area, cell 2. The global information around a single cell
shape, nucleo-cytoplasmic ratio, etc. Area is cluster
calculated by measuring the number of pixels 3. Information regarding the global connectivity
occupied by the cell. Similarly the maximum of the graph
number of pixels in the particular axis repre- 4. Information from the spectral graph
sents the maximum diameter of the cell (consid-
ering that the cell is round). Eccentricity is Data Fusion The image analysis data can be
calculated as dividing the minor axis length by combined with the epigenetic, genetic and pro-
major axis length. The nuclear shape is described teomic data. These complex data sets represent-
by circularity, eccentricity and irregularity of ing the spatial arrangement of the histopathology
the nuclei. These shape features are important to images, cell morphological features and genetic/
identify malignant cells. epigenetic information may be very helpful in the
disease classification, the grading of the tumor
Texture The texture analysis of the image of and the recognition of the aggressive subset of
interest gives important visual clues. Nuclear tex- the disease [15] (Fig. 18.3).
ture analysis helps in the assessment of interchro-
mosomal coarseness. The most commonly used Subcellular Quantification of the Substances
statistical method of texture analysis is grey level The computer-assisted digital image analysis is
co-occurrence of matrix (GLCM). The following able to quantify the immunohistochemically or
parameters describe GLCM: immunoflurescently stained cellular target pro-
teins on the slide or in tissue microarray speci-
• Energy men [16]. With the help of using colour code,
• Uniformity size and shape of the positively stained nuclei,
• Entropy the digitally scanned slide can be quantified for
• Smoothness particular substances. Various algorithms are
used to quantify the target proteins such as HER
Densitometric Features The various densito- 2 receptors, oestrogen and progesterone receptor
metric features include minimum grey value, (ER/PR), etc. Various commercially produced
maximum grey value, average grey value, mean softwares including hardwares are now available
grey intensity, standard deviation, kurtosis and to quantify the cytoplasmic, nuclear and mem-
the span of histogram. branous staining of the protein [9]. Automated
detection of fluorescent in situ hybridization
Pattern Recognition There are significant (FISH) is also possible with the help of computer-
advances in image analysis as whole slide scanning assisted image analysis technology [9].
190 18 Digital Image Analysis and Virtual Microscopy in Pathology
Data fusion
Disease classification
Prognostic prediction
Therapy guidance
18.5 T
he Current Problems
of Digital Image Analysis Box 18.2: Limitations and Difficulties of
Image Analysis
The various problems in DIA are described below • Auto-segmentation: Various modules
(Box 18.2): used such as edge-based, region-based
and model-based.
• Auto-segmentation: As mentioned before till • Decision on individual patient:
now, auto-segmentation unaided by any Knowledge-based software helps in this
human interference is the major challenge in aspect.
digital image analysis. The digital analysis • Three-dimensional imaging data.
system should be capable to identify the • Computational complexity and immedi-
images of interest. It should also process the ate application: Complex data set and
image and finally extract the data from the difficult to handle.
images. Different modules have been used to
solve the problem such as edge-based, region-
based and model-based module. • Three-dimensional imaging data: We gather
• Decision on individual patient: It is very two-dimensional image data which is too
important to take the decision of diagnosis or some extent not the real representation of the
classification or grading, etc. in individual cell. Three-dimensional construction of the
patients. Simple linear statistics is not helpful image and analysis of the data are the most
to take decision on individual patients. informative. With the help of the 3D recon-
Different knowledge-based techniques have struction, the ultrastructure of the cells is also
been used to solve this problem. The com- better studied [17].
monly used knowledge-based techniques are • Computational complexity and immediate
(1) K-means and spectral clustering, (2) artifi- application: The large amount of data gener-
cial neural network, and (3) supervector ated by the image analyser gives significant
machine. computational complexity and this may
18.6 Virtual Slide and Web-Based Teaching 191
19.1 Introduction
Conventiona Liquid based
The liquid-based cytology (LBC) is an increas- preparation preparation
ingly popular technique of the preparation of the
cervical sample and also other exfoliative sam-
ples such as effusion fluid, sputum, bronchoal-
veolar lavage, etc. This technique is relatively
costly than the other sample collection and prep-
aration. However, LBC preparation provides
clean, monolayered smear in small area of the
slide. As the smear is free of blood, mucus and
drying artefact, so it is easy to interpret [1, 2].
• Majority of the collected cells are available in preparation, and the cells are present in small
the liquid medium of the collection vial, area which is easy to screen (Fig. 19.1).
whereas the major part of the collected cells is • Monolayered preparation of the cells is pres-
sticked in the spatula of the convention smear ent in certain LBC preparation.
preparation and the cells are thrown in the • HPV test is possible for the residual material
waste basket. of LBC sample.
• LBC preparation is completely free of any air- • The monolayered cell preparation may be use-
drying artefact. ful in automated detection of malignant cells
• There is almost complete absence of any in the smear.
blood, mucus or necrotic debris in LBC
Negative suction
Rapid movement
removes the fluid
of the filter causes
and cells are
cellular dispersion
collected
on the filter
surface Cells are finally transferred
on the glass slide
Fig. 19.3 Schematic
diagram highlighting the
basic principle of
SurePath technique. The
cells are dispersed by
vortexing. Cell-rich
suspension is made by
buffy coat preparation.
These cells are taken out
and resuspended in fluid.
Finally the cells are
settled down by gravity
on the glass slide Vortexing
causes cell
dispersion Centrifugation
and enrichment of cells
a utomatically transfers the cells from the sur- 19.2.2 SurePath Test (Fig. 19.3)
face of the vial to the glass slide. The glass slide
is automatically submerged in the fixative The overall steps of SurePath test are highlighted
solution. in Fig. 19.3.
196 19 Liquid-Based Cytology and Automated Screening Devices in Cytology Sample
Fig. 19.4 Vortexing causes cell dispersion in SurePath Fig. 19.6 The vials are kept in the settling chamber to
technique settle the cells by gravity
Cell sedimentation
Table 19.1 Comparison of Pap test and SurePath • The system then scans the slides both in low
techniques and high power.
ThinPrep SurePath • The slides are ranked according to the degree
Methanol-based Ethanol-based collection of abnormality, and the device gives a score to
collection fluid fluid
each slide depending on the probability of
Completely automated Manual
abnormality.
Cell dispersion is done Cell dispersion is done by
by circular rotatory manual vortexing • The slide profiler classifies the slide as:
filter submerged within –– No further review: Highest probability that
the vial the smears are normal.
Cells are concentrated Cells are concentrated by –– Further review: Here manual review of the
on the surface of the density gradient, and
vial by negative suction therefore the cells may be
slide is needed to confirm the abnormality.
present in more than one Usually 75% of such cases contain abnor-
layer mal cells.
Blood and mucus may Better cellularity as there is
block the pores of the such problem BD FocalPoint GS Review Station This is a
filter
review station and the cytotechnologists followed
Good monolayered cell Cells are in multiple layers
preparation possible by cytologist review all the slides to detect the
abnormality.
Table 19.2 Differences of BD FocalPoint GS Imaging Table 19.3 Comparison of the automated screening ver-
System versus Hologic ThinPrep Imaging System sus manual screening
BD FocalPoint GS Imaging Hologic ThinPrep Automated
System Imaging System screening Manual screening
Special slide preparation is It is mandatory to have No chance of Tiring and boring job
not mandatory ThinPrep processing fatigue
and stain Automated system Erratic and subjective approach.
The cellular features The cellular features follows consistent Inexperienced worker may miss
mainly nuclear size, shape, along with optical logic the abnormal cells
nucleocytoplasmic ratio, density of the nuclei are Machine takes There may be variable time
etc. are picked up and analysed fixed amount of period of screening and
analysed time technologist takes longer time
The slides are ranked The most abnormal 22 Very costly device Cheap
according to “device score” fields are recorded in
and labelled as “review” or each slide. No ranks to
“no review” the slides are given The study group raised doubts about the imple-
Cytotechnologist can Cytotechnologist has to mentation of the automated screening devices
screen only “review” screen every slides
because of the reduced sensitivity and cost-
labelled slides again in those 22
marked areas effectiveness. There is no doubt that automated
screening devices increase the productivity of
• Place the slides in the cartridge that contains the cytotechnologists as less number of fields
25 slides. The total of 10 such cartridges can are needed to screen by them. Automation may
be operated at a single time. reduce the load of the tedious job of the cyto-
• The slides within the cartridge are imaged. technologists (Table 19.3). However, the
• The system records 22 FOV of the micro- increased economic burden in the screening
scopic fields that may contain abnormal cells. program may be one of the major obstacles of
• It transforms the information to the review the automation. Moreover, the HPV testing
scope along with manual screening may give more
meaningful result. Therefore, in the future we
Review Scope Place the slide in the review may have to give a serious thought before we
scope. implement automated screening.
g ynecologic cytology screening. Cancer Cytopathol. 8. Chivukula M, Saad RS, Elishaev E, White S, Mauser
2013;121(4):189–96. N, Dabbs DJ. Introduction of the Thin Prep Imaging
6. Passamonti B, Bulletti S, Camilli M, D’Amico MR, System (TIS): experience in a high volume academic
Di Dato E, Gustinucci D, Martinelli N, Malaspina practice. Cytojournal. 2007;4:6.
M, Spita N. Evaluation of the FocalPoint GS sys- 9. Kitchener HC, Blanks R, Cubie H, Desai M, Dunn G,
tem performance in an Italian population-based Legood R, Gray A, Sadique Z, Moss S, MAVARIC
screening of cervical abnormalities. Acta Cytol. Trial Study Group. MAVARIC - a comparison of
2007;51(6):865–71. automation-assisted and manual cervical screening:
7. Dawson AE. Can we change the way we a randomised controlled trial. Health Technol Assess.
screen?: the ThinPrep Imaging System. Cancer. 2011;15(3):iii–iv, ix–xi, 1–170.
2004;102(6):340–4.
Polymerase Chain Reaction:
Principle, Technique 20
and Applications in Pathology
Polymerase chain reaction (PCR) is one of the The basic steps of PCR are described as [1, 2]
most important techniques in molecular pathol- (Box 20.1, Fig. 20.1):
ogy [1, 2]. With the help of PCR, the single or the
pieces of target DNA can be amplified many
folds. This technique is now an integral part in Box 20.1: Principle of Polymerase
every modern laboratory for both the diagnosis Chain Reaction
and research use. The specific gene is amplified by using a
pair of DNA primer, heat-resistant DNA
polymerase enzyme and nucleotides.
20.1.1 What Is PCR and How It
Works? Steps
• Denaturation: Heat breaks the double-
PCR acts like a “molecular photocopying” stranded DNA to single-stranded DNA.
machine and amplifies the specific target DNA. • Annealing: Forward and reverse DNA
The basic principles of PCR are: primer bind with the complementary
DNA strand in 3′ region of each sepa-
• Double-stranded target DNA is made into rated DNA strand.
single-stranded DNA by applying heat. • Extension: Heat-resistant DNA poly-
• Two oligonucleotide strands or primers are merase (Taq polymerase) enzyme grabs
added. The oligonucleotide strand binds with the nucleotides and extends the DNA
its complimentary DNA strand to the 3′ ends. strand from 3′ to 5′ direction.
• The DNA strand is now extended with the
help of DNA polymerase (Taq polymerase). Repetition of this thermal cycle for
This polymerase enzyme incorporates the 25–30 times increases the DNA product.
nucleotides in the DNA to make it elongated.
• The cycle is repeated.
• Target DNA: 1 µLDNA template (1 ng total Cycling Time The PCR thermal cycle rapidly
amount) heats and cools the PCR reagent mixture. The
• Forward primer: 1 µL of 50 µM primer (final cycling time depends on (1) size of the DNA tem-
concentration is 1 µM) plate and (2) G-C content of DNA. The number
• Reverse primer: 1 µL of 50 µM primer (final of the thermal cycler is usually set as 25–30 cycles.
concentration is 1 µM) If the thermal cycle is increased more than 35,
• dNTPS: 4 µL dNTP mix ( 2.5 mM dNTP, final then too many unwanted DNA products may be
concentration is 200 µM) produced.
• Taq DNA polymerase: 0.25 µL of 5 U/µL of
Taq polymerase (total amount 1.25 U) The product is calculated as
• Buffer: 5 µL 10 X polymerase buffer (com-
M f = M ´ 2N
monly this buffer is supplied by the
manufacturer) Mf = final number of DNA molecule, M = initial
• MgCl2: 5 µL of 25 mM MgCl2 number of DNA molecule, N = number of PCR
cycle
Remember
Purification of the Amplified Product
• Add the template DNA and DNA polymerase The following measures are taken to purify the
just before the PCR to start. PCR products from the reaction solution:
• Please put at least one negative control and if
possible one positive control. • Agarose gel electrophoresis of the product:
Agarose gel electrophoresis is done from the
20.3.3 Thermal Cycling portion of the PCR product to verify the valid-
ity of the test.
• Close the cap of the PCR tubes and then put • Note the following things:
them in the thermal cycler. –– Any band present in the agarose gel elec-
trophoresis or not?
Standard Steps –– Is there any other bands of different
sizes?
• Initial denaturation: At 94 °C for 1 minute –– Is there smear pattern?
• Denaturation: At 94 °C for 30 seconds –– The successful PCR amplification product
• Annealing: 50–60 °C for 30 seconds: The shows a single sharp band with expected
temperature may vary depending on the size.
primer used. The temperature of annealing • Cloning of products: In this technique further
stage should be 3 to 4 °C lower than the melt- PCR is done to confirm the PCR product. This
ing point (Tm) of the primers. is done when the gene is present in very tiny
amount.
Tm of the primers = ( 4 ´ [G + C ]) + • Sequencing of products: This is done by auto-
mated sequencer machine to analyse the
( 2 ´ [ A + T ])
°
C
sequence of DNA formed as PCR product.
204 20 Polymerase Chain Reaction: Principle, Technique and Applications in Pathology
• Too less stringency in PCR: Too less strin- RNA of the target sample. This cDNA is then
gent condition generates unwanted DNA amplified by PCR technique [5].
products in PCR. 3. Asymmetric PCR: In this technique unequal
• Too much DNA template: Too much DNA concentration of primers is used. The great
template may produce undesired product in excess of primers is used for the targeted DNA
PCR. strand that we need to amplify. As the reaction
• Too many thermal cycles: In such case, proceeds, only the adequate amount of primer
reduce the number of thermal cycle 5–10. in the reaction mixture produces the particular
• Magnesium concentration is very high: DNA strand in excess. Therefore ultimately
Adjust the concentration and make it low. single-stranded DNA (ssDNA) is formed as
• Faulty primer: Redesign the primer. PCR product. As the reaction is slow and goes
• Carry over contamination: Change the on arithmetically, so many more cycles are
place of PCR. needed in this technique. Asymmetric PCR is
used for DNA sequencing and hybridization as
only one strand is needed in such conditions [6].
20.3.5 Enhancing PCR Products
Formation Disadvantages or limitations:
• The ssDNA is vulnerable to damage by
The following measures help to enhance the PCR many physical and chemical factors, and
products [4]: a more stable second structure may
form.
• Addition of non-ionic detergents: Triton • Different ssDNA may be formed even in
X-100, Tween 20, and NP-40 help to stabilize the same reaction.
the DNA polymerase enzyme and enhance the • It needs more thermal cycle.
reaction. However, these agents may lower the 4. Hot start PCR [7]: Normally DNA poly-
PCR stringency, and undesirable DNA prod- merase acts in the room temperature and even
ucts may be formed. More than 1% concentra- in the ice pack. Thereby there always remains
tion of these detergents may have inhibitory the possibility of spurious products. In hot
effect on PCR. start PCR technique, the DNA polymerase is
• The addition of dimethylsulfoxide (DMSO) in unreactive at the lower temperature and works
the G-C-rich DNA template (1–2%): Addition only at higher temperature. This is done by
of DMSO disrupts the base pair and enhances conjugating an inhibitor with the polymerase
the reaction. enzyme, and in the higher temperature, the
• Optimized annealing temperature: It is neces- inhibitor is free from the polymerase enzyme
sary to optimize the annealing temperature to and allows it to work.
increase PCR products. The various ways to do hot start PCR:
• Withheld the key agents until the end of ini-
tial denaturation process.
20.4 Types of PCR –– DNA polymerase enzyme or magnesium
cofactor
There are different types of PCR methods for • Mechanical barriers of the reagents:
diagnostic purposes. These are: –– DNA polymerase is encapsulated and is
only released at higher temperature.
1. Direct PCR: This is the standard PCR tech- –– Wax barrier is used to separate the key
nique as has been described in the previous components till the temperature is high.
section. –– Microfluidic devices are used to create
2. Reverse transcriptase PCR (RT-PCR): In case barrier.
of RT-PCR, at first cDNA is prepared from • Modification of DNA polymerase:
206 20 Polymerase Chain Reaction: Principle, Technique and Applications in Pathology
use a “TaqMan” probe. This is an oligonu- • Dual hybridization (Fig. 20.6): In this tech-
cleotide probe which is attached with a nique two hybridization probes are applied.
fluorescence reporter dye at its 5′ terminal The first probe is attached with a donor
and a quencher dye at the 3′ terminal end. fluorophore at the 3′ end, and the other
In case of the proper target sequence, this probe carries an acceptor fluorophore at the
probe anneals with one of the target 5′ terminal. In denaturation step there is no
sequence of DNA template. The “TaqMan” emission of fluorescence as any fluorescent
probe is cleaved by Taq polymerase during emission by donor fluorophore is degraded
PCR. When the “TaqMan” probe is intact, by the acceptor fluorophore. In the anneal-
the reporter dye and the quencher dye ing stage, the donor and the acceptor fluo-
remain in close proximity, and therefore rophore probes hybridize to the target DNA
fluorescence emitted from the reporter dye sequence, and they are adjusted in head to
is absorbed by the closely placed quencher tail position so that donor fluorophore
dye. So no fluorescence emitted. During comes in close contact with the acceptor
PCR the endonuclease breaks down the fluorophore. This allows fluorescence reso-
“TaqMan” probe, and the reporter dye is nance energy transfer. The intensity of the
away from quencher dye that allows emis- fluorescence is measured which is directly
sion of fluorescence. The increased inten- proportional to the amount of PCR
sity of fluorescence is directly proportional products.
to the amount of PCR products. • Molecular beacons (Fig. 20.7): In this
• DNA-binding dye (Fig. 20.5): In this tech- case the hybridized probe is designed like
nique DNA-intercalating agents SYBR®
Green are used. The SYBR® Green dye
molecules do not exhibit any fluorescence
in solution. However, the dye molecules
emit fluorescence when they are interca-
lated within the double-stranded DNA that
is formed after the primer extension and
polymerization. After each cycle the emit-
ted fluorescence from the polymerized
DNA is measured to estimate the total
amount of the amplified DNA.
Fig. 20.7 Schematic diagram shows the basic principle Fig. 20.8 Schematic diagram shows the basic principle
of molecular beacon technique to quantitate the PCR of nested PCR. Here more than two pairs of primers are
products. Here the hybridized probe is designed like a used for DNA amplification. At first primer is used for the
hairpin-like loop, and the reporter and quenching dyes are conventional PCR of the sample. In secondary PCR the
attached in the two ends of the loop. The close proximity product of the first PCR is used as the target of the second
of them prevents the emission of any fluorescence. During set of primers
annealing of the hybridized probe, the hairpin loop
becomes a straight probe, and the reporter and quenching
dyes stay away that allows emission of fluorescence
Table 20.2 Applications of PCR in basic research and (e) Forensic pathology: PCR technique is help-
clinical field ful in forensic pathology in different ways:
Applications of PCR • To detect paternity of the child
Basic research Clinical applications • To identity of the corpse or mutilated
• DNA • Diagnosis of infections body
sequencing
• To identify the criminal from the crime
• Bioinformatics • Cancer
• Classification – Detection of chromosomal site and biological materials of the
of organisms abnormalities criminal.
• Gene – Genetic mutation (f) Gene therapy: PCR helps to engineer the
expression – Detection of minimal residual specific gene to introduce in the diseased
studies disease
person to cure various diseases [20].
• Drug discovery • Genetic disease: intranatal
detection of inherited genetic
disease, e.g. Down’s syndrome,
Gaucher’s disease, etc.
• Forensic pathology: References
– Paternity detection
– Identification of mutilated 1. O’Leary JJ, Engels K, Dada MA. The poly-
body merase chain reaction in pathology. J Clin Pathol.
– Crime site investigation 1997;50(10):805–10.
• Gene therapy 2. Pan LX, Diss TC, Isaacson PG. The polymerase
chain reaction in histopathology. Histopathology.
1995;26(3):201–17.
3. Lorenz TC. Polymerase chain reaction: basic protocol
tumor suppressor gene such as mutation plus troubleshooting and optimization strategies. J Vis
in p53, c-myc, ras gene, etc. [17, 18]. Exp. 2012;63:e3998.
• Chromosomal changes: PCR helps to 4. Grunenwald H. Optimization of polymerase chain
reactions. Methods Mol Biol. 2003;226:89–100.
identify the specific chromosomal
5. Tse WT, Forget BG. Reverse transcriptase and direct
changes such as chromosomal transloca- amplification of cellular RNA transcripts by Taq poly-
tion, gene rearrangement, loss of hetero- merase. Gene. 1990;88:293–6.
zygosity, etc. [16]. 6. Gyllensten UB, Erlich HA. Generation of single
stranded DNA by the polymerase chain reaction and
• Monoclonality detection: PCR can detect
its application to direct sequencing of the HLA-DQA
B and T cell gene rearrangement and locus. Proc Natl Acad Sci U S A. 1988;85:7652–6.
thereby can prove the monoclonality in 7. Paul N, Shum J, Le T. Hot start PCR. Methods Mol
doubtful case of lymphoma [17]. Biol. 2010;630:301.
8. O’Leary JJ, Chetty R, Graham AK, McGee JO’D. In
• Minimal residual disease: In case of fol-
situ PCR: pathologist’s dream or nightmare? J Pathol.
low-up of a cancer case, PCR particularly 1996;178:11–20.
Q-PCR can detect and quantitate certain 9. Jong AY, T’ang A, Liu DP, Huang SH. Inverse
genetic change to detect any minimal PCR. Genomic DNA cloning. Methods Mol Biol.
2002;192:301–7.
residual disease of a patient [18].
10. Orita M, Iwahana H, Kanazawa H, Hayashi K,
( d) Genetic diseases: PCR technique is very Sekiya T. Detection of polymorphisms of human
helpful to detect various genetic diseases DNA by gel electrophoresis as single-strand confor-
such as Down’s syndrome, cystic fibrosis, mation polymorphisms. Proc Natl Acad Sci U S A.
1989;86:2766–70.
Gaucherec disease, etc. The main advantage
11. Dong Y, Zhu H. Single-strand conformational poly-
of PCR technique is that it can bypass the morphism analysis: basic principles and routine prac-
aggressive placental bed biopsy to detect tice. Methods Mol Med. 2005;108:149–57.
these inherited diseases. The minute amount 12.
Higuchi R, Dollinger G, Walsh PS, Griffith
R. Simultaneous amplification and detection of specific
of foetal cells collected from the mother’s
DNA-sequences. Biotechnology. 1992;10(4):413–7.
blood or cervical mucosa are enough to reach 13. Arya M, Shergill IS, Williamson M, Gommersall
at a diagnosis [19]. L, Arya N, Patel HR. Basic principles of
References 211
real-time quantitative PCR. Expert Rev Mol Diagn. 18. Lee MS, Chang KS, Cabanillas F, Freireich EJ,
2005;5(2):209–19. Trujillo JM, Stass SA. Detection of minimal residual
14. Greiner TC. Polymerase chain reaction: uses and
cells carrying the T(14:18) by DNA sequence amplifi-
potential applications in cytology. Diagn Cytopathol. cation. Science. 1987;237:175–8.
1992;8(1):61–4. 19. Tutschek B, Sherlock J, Halder A, Delhanty J, Rodeck
15. Bermingham N, Luettich K. Polymerase chain
C, Adinolfi M. Isolation of fetal cells from transcer-
reaction and its applications. Curr Diagn Pathol. vical samples by micromanipulation: molecular con-
2003;9(3):159–64. firmation of their fetal origin and diagnosis of fetal
16. Ronai Z, Yakubovskaya M. PCR in clinical diagnosis. aneuploidy. Prenat Diagn. 1995;15:951–60.
J Clin Lab Anal. 1995;9(4):269–83. 20. Sun H, Pan Y, He B, Deng Q, Li R, Xu Y, Chen J, Gao
17.
Wan JH, Trainor KJ, Brisco MJ, Morley T, Ying H, Wang F, Liu X, Wang S. Gene therapy for
AA. Monoclonality in B cell lymphoma detected in human colorectal cancer cell lines with recombinant
paraffin wax embedded sections using the polymerase adenovirus 5 based on loss of the insulin-like growth
chain reaction. J Clin Pathol. 1990;43:888–90. factor 2 imprinting. Int J Oncol. 2015;46(4):1759–67.
Fluorescent In Situ Hybridization
Techniques in Pathology: 21
Principle, Technique
and Applications
• Dehydrate the smear by dipping in 70%, 7. Visualization: If the probes are directly
80% and 95% ethyl alcohol and dry the labelled with the fluorochrome dye, then no
smear. further procedure is needed. In that case,
• Treat the smear with proteinase K solution counterstain the slide by 5 μl DAPI/antifade
(20 μg/ml) for 15 min in room temperature. solution. Now visualize the cells by an epiflu-
• (Proteinase K solution preparation: add 32 μl orescence fluorescence microscope.
of proteinase K solution (25 mg/ml proteinase
K solution) in 40 ml 2× SSC, pH 7.4.)
• Gently wash the slide in deionized water. 21.3 Troubleshooting
• Dehydrate the smear.
Saline sodium citrate (SSC): Table 21.1 has described the problems in FISH
Add techniques and their remedies.
Sodium chloride 175.3 g
Sodium citrate 88.2 g
Distilled water 800 ml 21.3.1 Different Types of FISH
Keep pH 7.2 by adding drops of 10 N solution of 1. Three-dimensional FISH (3D FISH): In this type
NaOH. Now add water and make it 1 litre. of FISH, multiple images of the nuclei are taken,
4. Denaturation: and with the help of a suitable software, a three-
Denaturing of target DNA: Denature DNA dimensional image is made [8]. 3D FISH helps
of the target cells in the smear by treating the to study the topology of the genes in respect to
smear with denaturing solution for 2 min time the chromosomal territory within the nucleus.
at 72 °C. 2. Living cell cytogenetics (four-dimensional
Denaturing solution: 70% formamide in 2× FISH): Fluorescent-tagged nucleotide can be
SSC (add 10 ml of double-distilled water, incorporated into DNA that may help in the
5 ml of 20× SSC, 25 μl of 250 mM EDTA simultaneous visualization of DNA distribution
and 35 ml of formamide). and genomic organization in the living cells [9].
Denaturing of probe DNA: Add 1 μl of 3. Multi-coloured FISH (M-FISH)/spectral
labelled probe with 9 μl of 65% for- karyotyping (SKY): In the case of SKY tech-
mamide solution, 10% dextran sulphate nique, DNA is tagged with different fluoro-
in 2× SSC. chrome dyes (the chromosome-specific
Now heat the mixture at 75 °C for painting probes), and all the chromosomes are
5–6 min. stained. The different chromosome therefore
5. Hybridization: takes different colour (Fig. 21.2). There are
• Add 10 μl of denatured probe solution over three essential steps of M-FISH:
the slide. (a) Hybridization: The fluorescent-tagged
• Put a coverslip over the smear and close the whole chromosome probes (WCP) are
margins of the coverslip. hybridized with the metaphase chromo-
• Incubate it at 37 °C for 1 day (24 h). some spread. WCPs consist of multiple
6. Post-hybridization: probes tagged with spectrally different
• After the incubation, remove the coverslip fluorochrome in combinatorial manner.
gently, and rinse the slides in SSC for (b) Visualization and image acquisition: In
5 min twice. this step the images of the chromosomes
• Put the slides in a Coplin jar filled with pre- are visualized by the fluorescence micro-
warmed SSC at 70 °C. scope with attached filters. Subsequently
• Keep the slide in Coplin jar filled with SSC the images are acquired by the digital
at room temperature. camera and appropriate software.
216 21 Fluorescent In Situ Hybridization Techniques in Pathology: Principle, Technique and Applications
(c) Analysis: Finally the images are analysed • Add 10 μl denatured M-FISH probe solution
with the help of the specialized software over the denatured chromosome preparation.
to find out any structural chromosomal • Put coverslip over the smear and seal the mar-
abnormalities. gins of the coverslip with rubber cement.
Steps of M-FISH [10, 11] • Keep the slide in a humidified chamber for
Pretreatment of the metaphase spread by 2 days at 37 °C.
trypsin: • Remove the slide from the chamber, and take out
• Incubate the slide in pepsin at 37 °C for the coverslip by removing the rubber cement.
5 min. • Wash the slides in SSC at 72 °C for 2 min.
• (Pepsin 1: 20,000 in 10 mM HCl.) • Counterstain with DAPI and place a
• Wash the slide in PBS: 5 min. coverslip.
• Fix the slide by 1% formaldehyde. • The slide is now ready to visualize in the fluo-
• Wash again in PBS: 5 min. rescence microscope.
• Dehydrate in serially graded alcohol (70%, 4. Comparative genomic hybridization (CGH):
90% and 100%) for 2 min each. CGH provides the global view of gain or loss
Denaturation of chromosome of chromosome of the tumor genome [12].
• Incubate the slide in 50 ml denaturing solution
within a Coplin jar for 3 min at 72 °C. Basic principles (Fig. 21.3):
• Immediately dip the slide in ice-cold ethanol Tumor DNA is extracted from the sample and
70%, 90% and 100% each for 2 min. labelled by green fluorochrome dye.
• Dry the slide. Normal DNA from the control is also extracted
and labelled by red fluorochrome dye.
Denaturation of probe The mixture of both green-labelled tumor DNA
• Centrifuge the M-FISH probes. and red-labelled control normal DNA is mixed
• Mix the contents gently and take 10 μl probe and allowed to hybridize on the normal meta-
solution for each slide in an Eppendorf phase chromosome.
Tube. With the help of digital image analyser, the green/
• Incubate the Eppendorf Tube at 80 °C for red ratio is measured.
5–7 min to denature the probe. Excess green fluorescence represents chromo-
Hybridization somal gain or amplification.
21.3 Troubleshooting 217
Fig. 21.3 Schematic
diagram shows the basic
principle of comparative
genomic hybridization.
Tumor DNA is labelled
by green fluorochrome
dye, and normal DNA
from the control is
labelled by red
fluorochrome dye. They
are allowed to hybridize
on the normal metaphase
chromosome. With the
help of digital image
analyser, the green/red
ratio is measured. The
excess green
fluorescence represents
chromosomal gain or
amplification, whereas
excess red fluorescence
represents chromosomal
loss
218 21 Fluorescent In Situ Hybridization Techniques in Pathology: Principle, Technique and Applications
Excess red fluorescence represents chromosomal 2. Add 1/10th volume of 3 M sodium acetate
loss. (pH 5.2) and mix them.
Advantages of CGH: CGH technique is helpful in 3. Add 2.5 volume of 100% ethyl alcohol (ice
tiny amount of micro-dissected tissue. The cold) and vortex again.
technique can be done without any prior 4. Remove the supernatant fluid.
knowledge of the chromosomal abnormalities 5. Resuspend the pellet by adding 10 μl hybrid-
in the tumor DNA. ization mixture.
Li mitations of CGH: CGH is not helpful if there
Denature target metaphase smear and hybridiza-
are chromosomal abnormalities without any
tion mixture
gain or loss of genetic material. Similarly
from CGH we do not get any information on 6. Fix the metaphase slide by 4% paraformal-
the structural changes of the chromosome. dehyde for 15 min at 4 °C.
CGH is much less sensitive than PCR, and 7. Rinse the slide in PBS.
the result may be changed due to contamina- 8. Incubate the slide in proteinase K solution
tion of normal cells with tumor cells. for 5 min at 37 °C.
9. Rinse in PBS for 5 min.
10. Denature the metaphase spread smear: Keep
Box 21.2 Advantages and limitations of CGH the slide in preheated Coplin jar in water
Advantages bath containing denaturing solution at 75 °C
for 5 min.
• Tiny micro-dissected tissue can be pro- 11. Dehydrate the slide by graded alcohol and
cessed for CGH. dry in air.
• No need of details of chromosomal 12. Simultaneously denature the DNA samples:
abnormalities of tumor tissue. Heat the sample at 75 °C for 5 min. Immediately
incubate the sample at 37 °C for 20 min.
Limitations
Hybridization
• Ineffective technique if there are chro- 13. Pour 10 μl of the probe mixture on the meta-
mosomal abnormalities without any phase smear. Cover the area by a coverslip
gain or loss of genetic material and seal the edges by rubber cement.
• No information of any structural 14. Incubate the slide in a humid chamber at
abnormalities 37 °C for 48 h.
• Tedious and prolonged process
Washing
Fig. 21.4 Schematic
diagram shows the basic
principle of array-based
comparative genomic
hybridization. Tumor
DNA is labelled with a
green fluorochrome dye,
and the control DNA is
labelled with red
fluorochrome dye. The
mixture of them is
hybridized with multiple
specific DNA probe in
the microarray plate, and
the hybridization
reaction plate is
analysed
220 21 Fluorescent In Situ Hybridization Techniques in Pathology: Principle, Technique and Applications
• At first make a list of suitable cases. Diameter of the Core The diameter of the
• Study all the section of these cases. punches of the core biopsy may vary from 0.6 to
• Review the cases and reclassify if needed. 2 mm. In fact most of the people take 0.6 mm
Then mark the representative area on the diameter core that includes 0.14 square mm
stained section on glass slide. It is preferable tissue.
to use different colour for different diagnostic
area such as green for normal area, red for Number of Cores in TMA In a 2.5 × 4.5 cm
tumor and black for in situ carcinoma. block, at least 1000 cores can be adjusted.
• Collect the representative block of the slides However it is preferable not to take more than
and identify the area. 500 cores in a single block.
Fig. 22.3 The
schematic diagram of
the organization of the A B
grid for tissue
microarray. The capital
letters indicate the
quadrant, and small
letters indicate the
co-ordinate of the tissues
in the recipient’s block.
The primary data for
each tissue core such as
biopsy number, location,
diagnosis, grade of
tumor, stage, etc. are
entered in a Microsoft
Excel file
C D
1. Amplification of the resource: Ordinarily from a 10 batches of 20 slides each), and the slide-to-
standard 5 mm thick section of tissue, we can slide variation may occur due to several vari-
get maximum 100 sections for study. Whereas ables such as antigen retrieval, concentration
depending on the size of the tissue in the origi- of different reagents, incubation period, wash-
nal block, at least 200 core biopsies can be taken ing time, etc. However in case of TMA, each
to make TMA block. After the construction of tissue section consists of 100–1000 core biop-
the TMA, we can cut at least 1000 sections of sies from the different patients, and the single
3 μ thickness from the 5 mm thick array block. section is stained that avoids all the slide-to-
Therefore we get a total 1000 × 200 means slide variability.
200,000 sections from the limited resource. 3 . Faster, cheaper and reduction of manpower:
2. Uniformity in staining conditions: At the time In TMA a single slide requires less reagents,
of conventional staining, the different tissue labour and time. Therefore, TMA saves cost,
sections are stained at different time (such as time and total work force.
224 22 Tissue Microarray in Pathology: Principal, Technique and Applications
Limitations:
• Tissue heterogeneity
• Not suitable in small series with occa-
sional use
Fig. 22.4 The schematic diagram shows the elaborated • Loss of the tissue
design of the grid for tissue microarray. The peripheral • Confusion on orientation
boundary walls of the TMA are made of single row of
core tissue. Positive and negative control tissues are
placed in asymmetric location (green dots). The tumor tis-
sues are placed in small groups (red dots). Normal healthy
tissues are placed in one or two rows (black dots) 22.5 Limitations of TMA
4. Original block can be preserved: Only a few The main limitation of TMA is tissue
core biopsies from the original block are suf- heterogeneity.
ficient to make TMA. The original block can
be preserved and can be used for further 1. Tissue heterogeneity: This is one of the main
sectioning. concerns of TMA. The tumor may vary from
5. Effective in quality assurance program: Due area to areas such as Hodgkin lymphoma
to significant amplification of the laboratory which may have different morphologies in
material, TMA can be used for external and different areas. Therefore small 2 mm tissue
internal quality control program. The TMA may not represent the whole tumor and find-
section can also be used for standardization of ing may vary. However, several studies have
reagents for positive control. shown that TMA and whole tissue data are
6. The whole analysis of TMA is now automated almost similar [5, 6].
and computer can keep track of the huge data. 2. Less cost-effective in small series of cases:
TMA is not very effective if it is done once in
a while in a small series of cases.
Box 22.1: Advantages and Limitations of 3. Prone to loss the tissue: The core biopsy tis-
TMA sues may be lost due to its tiny size. Tissue
rich in keratin, bone or cartilages are likely to
Advantages: be lost.
4. Disorientation of the core biopsy tissue: Due
• Amplification of the resource: Nearly to large number of core biopsies, there is a
about 100,000 times amplification is chance of disorientation when TMA is done
possible. manually. Rows of empty core tissues may
• Uniformity in staining conditions: help in the immediate orientation of the
Overall uniformity of the staining is tissue.
feasible.
• Faster and cheaper: When large number
of core tissue is processed, it becomes
cheaper.
References 225
22.6 Clinical Applications of TMA 2. Dancau AM, Simon R, Mirlacher M, Sauter G. Tissue
microarrays. Methods Mol Biol. 2016;1381:
53–65.
TMA helps to implement various molecular dis- 3. Bubendorf L, Nocito A, Moch H, Sauter G. Tissue
coveries in clinical areas. The novel genes can be microarray (TMA) technology: miniaturized pathol-
identified by DNA microarray technique, and ogy archives for high-throughput in situ studies. J
Pathol. 2001;195(1):72–9.
subsequently these gene products can be vali- 4. Parsons M, Grabsch H. How to make tissue microar-
dated by TMA [7, 8]. rays. Diagn Histopathol. 2009;15:142–50.
There are three different categories of TMA 5. Kononen J, Bubendorf L, Kallioniemi A, et al.
which are described in this respect: Tissue microarrays for high-throughput molecu-
lar profiling of hundreds of specimens. Nat Med.
1998;4:844–7.
1. Different types of tumor for the novel markers: 6. Parker RL, Huntsman DG, Lesack DW, Cupples
Large varieties of tumor can be studied for the JB, Grant DR, Akbari M, Gilks CB. Assessment of
novel markers in an ultrashort time. interlaboratory variation in the immunohistochemi-
cal determination of estrogen receptor status using
2. Tumor progression array: One particular a breast cancer tissue microarray. Am J Clin Pathol.
tumor type is studied for its progression such 2002;117(5):723–8.
as in a single section, one can study normal 7. Sallinen S-L, Sallinen PK, Haapasalo HK, et al.
prostatic tissue, prostatic hyperplasia, intraep- Identification of differentially expressed genes in
human gliomas by DNA microarray and tissue chip
ithelial neoplasia and carcinoma [9]. techniques. Cancer Res. 2000;60:6617–22.
3. Prognostic array: Novel markers related to 8. Moch H, Schraml P, Bubendorf L, et al. High-
the prognosis of the malignant tumor can be throughput tissue microarray analysis to evaluate
studied by TMA. genes uncovered by cDNA microarray screening in
renal carcinoma. Am J Pathol. 1999;154:981–6.
9. Varambally S, Dhanasekaran SM, Zhou M, Barrette
TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta
References KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan
AM. The polycomb group protein EZH2 is
1. Fejzo MS, Slamon DJ. Frozen tumor tissue microar- involved in progression of prostate cancer. Nature.
ray technology for analysis of tumor RNA, DNA, and 2002;419(6907):572–3.
proteins. Am J Pathol. 2001;159:1645–50.
Sanger Sequencing and Next-
Generation Gene Sequencing: 23
Basic Principles and Applications
in Pathology
Advantages:
Limitation:
Fig. 23.3 Schematic
diagram showing the
basic principle of
pyrosequencing
technique. Here fixed
amount of inorganic
pyrophosphate (PPi) is
released whenever a
nucleotide is
incorporated in
polymerization reaction
by DNA polymerase
enzyme. The released
PPi initiates a chain of
reaction that liberates
light energy and is
detected by colour-
charged device camera.
The DNA sequence is
assessed from the
pyrogram that is
generated during each
nucleotide incorporation
integrated sample processing, DNA amplifi- obtained. Finally the complete DNA sequence is
cation, isolation and concentration of DNA decided by assembling the overall information
fragments and sequencing. from the multiple hybridization tests.
There are two ways to hybridize: (a) The DNA
Advantages: is immobilized on a membrane, and then various
small oligonucleotide probes are used for hybrid-
• Very fast technique ization. (b) Microfabricated tilling array contains
• Minimal reagent consumption more than 6,000,000 distinct probes, and the
• Good quality optical property genomic DNA to be sequenced is hybridized to
determine the complete base sequence of the
entire DNA [5].
3. Hybridization sequencing (Fig. 23.4): In this
technique, numerous oligonucleotide probes are Advantages:
used to hybridize with the target DNA. The com-
plementary probes are hybridized with DNA, • Fast
and therefore the sequence of bases of DNA is • High throughput
230 23 Sanger Sequencing and Next-Generation Gene Sequencing
23.2.2 Limitations
24.1 Light
Hue Saturation Intensity (HSI) This is one Red Green Blue (RGB) There are three pri-
of the common modules to express colour. Hue mary colours: red, green and blue. The other
indicates the basic colour such as blue, green or colours that we perceive are the mixture of these
red. The saturation means how deep is the colours such as admixture of red and green colour
colour that means whether the colour is pale or will produce yellow colour. The percentage
Ultraviolet Retina
Infrared Fovea
Lens
Image
Cornea
Object
Visible light
Iris Optic
400 nm 750 nm
Sclera nerve
Fig. 24.2 Visible spectrum of light is 400–750 nm wave- Vitreous humor
length of light
Fig. 24.3 Image formation in the human eye is high-
expression of these three primary colours repre- lighted in this schematic diagram. The image of the object
is formed on the retina of the eye. The initial image in the
sents the colour of that particular object. retina is an inverted image of the object. The photorecep-
tor cells of the retina pass the electrical signal through the
CYM The subtraction of one or more primary optic nerve to the brain. The human brain corrects the
colour from the white light produces three other inverted image to the normal erect image
secondary colours: cyan (C), yellow (Y) and
magenta (M). Therefore this CYM colour model
is known as primary subtractive colours. This
model is mainly used in optical filter and
printing.
lens and the focal centre where the image is per- them as two separate entities. However, the more
fectly focused. and more they are kept close together, the more it
is difficult to recognize them as two separate enti-
Light Microscope [1] The light microscope ties. At a certain distance, it may be impossible to
deals with the visible light and so it is named as distinguish these two objects, and they may look
light microscope. The predominate three func- as one object. The minimum distance where the
tions of this microscope are: two objects can be distinguished separately is
known as resolution capacity (D) of the micro-
• Magnification
scope. The resolution of the microscope is depen-
• Resolution
dent on two factors:
• Contrast
1 . The wavelength of the light
Magnification The word magnification indi-
2. The maximum angle of light that can be
cates the enlargement of the image of object of
obtained by the objective lens from the object
interest. The objective and the eyepiece take part
in the magnification of the image of the object.
Numerical Aperture (Fig. 24.5) Numerical
The first magnification takes place by the objec- aperture (NA) is related with the light gathering
tive. The power of magnification is written on the power of the objective. In fact the resolution
wall of the objectives of the microscope. power of the microscope is largely dependent on
Normally the power of magnification varies from the NA of the objective, and it is represented by
4 times to 100 times in ordinary biological light the equation below:
microscope. The second magnification is done by
the eyepiece, and so the final magnification is 0.61s
D=
equal to the first magnification done by objective NA
multiplied by the second magnification done by
the eyepiece. D = resolution
Sigma (σ) = the wavelength of light
M = M1 ´ M 2 NA = numerical aperture
Numerical aperture is calculated as:
M = Final magnification NA = n × Sin μ
M1 = Linear magnification by objectives n = refractive index of air (it is 1)
M2 = Linear magnification by eyepiece μ = half of the angular aperture of the
The final magnification of the different objec- objective
tives is shown in Table 24.1.
24.5.1 The Major Aberrations lens with different dispersing properties for the
of the Lens different colours so that the final images overlap
in the same location.
The spherical lens of the microscope may have
several optical faults or aberrations. Image dis- Spherical Aberration (Fig. 24.9b) The spheri-
tortion occurs due to these various optical aberra- cal aberrations occur when spherical lens is used
tions. The common aberrations are: for magnification in the microscope. The cause of
spherical aberrations is the different bending
Chromatic Aberration (Fig. 24.9a) The chro- capacities of the central and peripheral part of the
matic aberration occurs due to the different spherical lens. The incident rays of light that pass
refractions of different wavelengths of light. Blue through the peripheral part of lens bend more
light is bended more than the red light. Therefore than the incident rays that pass through the centre
there are different locations of images for the dif- of the lens. Therefore in case of spherical aberra-
ferent wavelengths of the colour, and overall the tion, the images are formed in different focal
image becomes distorted. More disastrous is the planes, and the image may be blurred in the
different magnifications of the different colours periphery.
of images as they are located in different dis-
tances from the lens. Solution: The solution of spherical aberrations
is to apply a collection of different thickness of
Solution: The solution of the chromatic aber- positive and negative lens.
ration is to use the different components of the
Astigmatism This is an off-axis aberration, and
a it occurs due to the defect in the manufacture of
the proper curvature of the lens or defective
placement of the lens. Here the rays of light from
the object going through the horizontal and verti-
cal diameters of the lens focus in two separate
focal planes. The astigmatism increases when the
object is more away from the optic axis.
24.7 Other Types of Microscope be kept at low. The light passes through the sam-
ple, and the denser area of the object absorbs part
24.7.1 Dark-Field Microscope of the light. So the object looked dark in a bright
background.
In this type of microscope, a hollow beam of light Use: Stained or natural specimen
is produced by blocking the central part of the Advantages:
beam of light (Fig. 24.10). The scattered light • Very simple.
from the object passes through the objective to • No additional equipment’s are needed.
the eye, and the object is seen as bright in a dark • Object can be seen without staining.
background. Ordinary light microscope can be Limitations:
used as dark-field microscope by using dark-field • Low contrast
block condenser. The central cylinder of light is • Low resolution
obstructed, whereas the peripheral rim of light • Low magnification
reaches to the object.
• The bacteria, spirochaetes, or fungi in suspen- Principle In this microscope the objects having
sion are better seen in dark-field microscope. different refractive indices are identified as they
• Movement of the cells in culture medium is produce different contrasts. The object with scat-
better seen in this microscope. tered light is identified from the illuminating back-
ground light. If the light passes through a transparent
object, then due to the change of refractive index of
24.7.2 Bright-Field Microscope the object, the pathway of light will be deviated
slightly, and the light wave is retarded. In case of
This is the simple light microscope where the denser particle (higher refractive index) in the
object is examined by attenuated light. No addi- object, the deviation of light will be more, and the
tional equipment is required for the bright-field light wave is more retarded. This is known as phase
microscopy. The diaphragm of the microscope difference. Usually these phase differences are
should be fully opened, and light intensity should invisible to us; however, the phase contrast micro-
scope makes these changes significantly visible.
Objective
System (Fig. 24.11) In phase contrast micro-
Divergent beam of
light
scope, a condenser annulus and modified objec-
tive with phase plate are used. Intense beam of
Object light source is passed through the substaged con-
Cone of light denser annulus which is located in the focal plane
of the condenser. So a hollow cone of light is gen-
Condenser
erated that can be controlled. Now this light
passes through the objects/sample, and they are
Light either deviated or undeviated depending on the
blocked refractive indices of the different structures of the
object. Now both undeviated (central ray) and
deviated light pass through the objective and are
Fig. 24.10 Schematic diagram showing pathway of light
segregated by the phase plate that is located
in the dark-field microscope. A hollow beam of light is
produced by blocking the central part of the beam of light behind the focal plane of the objective.
References 243
Applications:
References
1. Wollman AJ, Nudd R, Hedlund EG, Leake MC. From
animaculum to single molecules: 300 years of the
light microscope. Open Biol. 2015;5(4):150019.
2. Wolf DE. The optics of microscope image formation.
Methods Cell Biol. 2003;72:11–43.
3. Goodwin PC. A primer on the fundamental prin-
ciples of light microscopy: optimizing magnifica-
tion, resolution, and contrast. Mol Reprod Dev.
2015;82(7–8):502–7.
Fluorescence microscopy applies high-inten- • The fluorescent dye absorbs high-energy light
sity light to illuminate the substance that emits and immediately releases photon of low-
fluorescence light. The light of shorter wave- energy light.
length strikes the fluorescent object, and the • The emitted light is passed through the filter
object emits the light of the longer wavelength, and is detected by the observer against a high-
and the image of the object is visualized by the contrast black background.
observer.
Fig. 25.1 Schematic
diagram showing the Excited state
principle of fluorescent
dye
Absorption
of photon
Emitted light
Excitation light
Fluorescence
or emission
Flourescence
light released
Ground state
Excitation
light Electron
Orbit
Fluorescent molecule
Eye
to the excitation maximum of the particular
fluorescent dye.
Eye piece
Object
25.2 Incident Fluorescent
Excitation Microscope
Red stop filter beam of light
In this type of microscope, a dichroic mirror is used
Heat Filter
(Fig. 25.3). This dichroic mirror has certain unique
properties. The mirror only allows the selected exci-
Light Selective tation beam of light to be reflected on the object,
source filter and the remainder unwanted beam of light is trav-
elled through the mirror and is lost. Similarly, the
Fig. 25.2 Schematic diagram showing the principle of emitted fluorescent light from the object is allowed
transmitted fluorescent microscope. The beam of light to pass through the mirror to the eyepiece. Therefore
passes through the multiple filters and ultimately hits the
object. The excitation beam passes through the objective in incident fluorescent microscope, the high-energy
towards the barrier filter and reaches to the observer light passes through the various filters, and then
25.3 Confocal Microscopy 247
Eye
Table 25.1 Maximum excitation and emission spectra of
different fluorochrome dyes
Eye piece Maximum Maximum
excitation emission
Fluorochrome dye wavelength (nm) wavelength (nm)
Barrier filter
Selective Fluorescein 494 520
filter Objective isothiocyanate
(FITC)
Dichroic Hoechst 33342 346 460
mirror DAPI 359 461
Rhodamine 570 590
Cy3 548 561
Light Texas Red 595 613
source Emitted
beam of light
Excitation • Hoechst 33342
beam of light
• Rhodamine
Object • Texas Red Cy3
collected and stored as signal. The signals are 5. Reconstruction of three-dimensional images
finally displayed in a video monitor. There is possible.
may be multiple PMT for the signal process- 6. Images are free from artefacts seen in ordi-
ing of simultaneously different fluorochrome nary microscope.
dyes.
5 . Computer with appropriate software: The
computer is an essential component of CFM 25.4 Limitations of CFM
and has the following functions: image detec-
tion, image processing, image reconstruc- CFM has the following limitations:
tion, image storage and video display of the
image. 1. Very expensive.
2. Photobleaching technique is not very good
Advantages (Box 25.1): compared to conventional microscope.
3. Depth of penetration of CFM in live image is
1. It helps to assess the spatial distribution of limited.
various intra- and extracellular macromole-
cules of the cells [3].
2. It provides high-resolution and high-contrast 25.5 Applications of CFM [5–7]
clear images.
3. CFM can work on living tissue, and the fixa- Table 25.2 highlights the biological applications
tion of tissue is not required for the study of of CFM.
confocal fluorescence imaging.
4 . Thick tissue section can be studied by
1. Study of fluorochrome-stained section: Single
CFM [4]. or multiple fluorescent dye-stained samples
can be studied by CFM. It can detect simulta-
neously many fluorochrome dyes. Moreover
fluorescence recovery after photobleaching
(FRAP) is possible in CFM.
2. The detection of co-localization: CFM is help-
ful to determine the exact localization of two
closely situated tissues. It provides blur-free
very good resolution of fluorescent-stained
sample.
3. Green fluorescence protein (GFP): CFM can
Fig. 25.6 Two types of scan occurs in confocal micro- help to track the distribution and function of
scope: line scan and frame scan
protein by tagging it with GFP. It helps in
tracking cell signalling pathway, intracellular
trafficking and also gene expression in the
Box 25.1: Advantages of Confocal cells.
Microscope 4. Epitope tagging: CFM is helpful in the track-
• Spatial distribution of intracellular and ing of epitope of the antigen by tagging it with
intercellular substances. green fluorescence protein (GFP).
• High-contrast, high-resolution images. 5. Diagnosis: Screening of the colorectal cancer
• Live images possible. can be done with the help of CFM, etc. It also
• 3D reconstruction. helps to measure the corneal thickening.
• Free from artefacts. 6. Functions of cytoplasmic organelles:
• Thick tissue can be studied. Organelle-specific fluorescent probes are
helpful to study the function of various cellu-
250 25 Fluorescence and Confocal Microscope: Basic Principles and Applications in Pathology
Absorption Two
of photon photons
Emitted light excitation Emitted light
Fluorescence Fluorescence
or emission or emission
λ is wavelength, h = 6.626 × 10−34 (Planck’s con- 1. Electrons have shorter wavelength and pro-
stant), m = mass and v = speed of the electron. vide very high-resolution capacity.
Now increasing the speed of the electron, we can 2. It is easy to manipulate.
reduce the wavelength significantly, and 0.001 nm 3. Electron gives high brightness.
wavelength of the electron can easily be achieved. 4. Electron beam interacts strongly with matter
Table 26.1 Comparison of electron microscope and cathode shield is negatively charged and deflects
light microscope the electron to make it a central beam. The central
Characteristic Light beam of electron emerges from the small hole of
features microscope Electron microscope the (Wehnelt cylinder) cathode shield.
Probe used Ordinary High-energy
visible light: electron beam: 4 nm
700–300 nm monochromatic Sample Illumination (Condenser System)
Maximum 0.5 μm 0.1 nm Several lenses are used as condenser for focusing
resolution the electron beam in a particular plane. Unlike
Maximum 1000 times 500,000 times light microscope, in case of electron microscope,
magnification we use electromagnetic coil as lenses. By apply-
Condenser Made of Electromagnetic coil
glass
ing electrical current through the coil, the strong
Objective Made of Electromagnetic coil magnetic field is created. The strength of the
glass magnet can be changed by adjusting the electri-
Interior of the Air filled Vacuum cal current through the coils. So if we increase
optic column the current, then the focal length of the beam will
Image On eye On the fluorescent be shortened, whereas, reducing the current will
formation screen
increase the focal length.
Microscopic column
Digitized image
in computer
Fluorescent screen
b Electron source
Anode plate
Sample
Objective lens
Screen
Fig. 26.2 (a) Electron microscope and its parts. (b) The various components of transmission electron microscope and
microscopic column have been highlighted in this schematic diagram
256 26 Electron Microscopy: Principle, Components, Optics and Specimen Processing
1. Elastic scattering: This is interaction between hit the atom of the specimen. The orbital electron
the nucleus of the atom and the electron of the gets excited and leaves the atomic orbit and
beam (primary electron). In this type of reac- moves towards the surface of the object. This
tion, the kinetic energy and velocity of the pri- released excited electron from the atom is known
mary electron are unaltered. Only the pathway as secondary electron. The secondary electron
of electron is altered. The nucleus of the atom also undergoes elastic and inelastic interaction
is very tiny (3 × 10–15 m) compared to the atom and ultimately exits from the surface. This sec-
as a whole (3 × 10–10 m). Therefore, there is ondary electron can be detected. This is the basis
actually minimal chance of truly hitting the of scanning electron microscope (SEM).
electron with the nucleus. However, the posi-
tive electrostatic force of the nucleus works on Backscattered Electrons When the high-
the electron and deflects it from its pathway. energy incident beam of electron hits the speci-
2. Non-elastic scattering: Here, the principle men, some of the electrons of the incident beam
electron of the microscope column interacts are reflected back towards the surface. These
with the electrons of the orbit of the atom. The electrons are known as backscattered electrons.
orbital electron repulses the incident principle The object with higher atomic number will have
electron. Here also the pathway of the electron more backscattered electrons than that of lower
is changed, and moreover the energy is lost by atomic number objects.
the principle electrons.
Excited Electrons of the Atom The incident
Secondary Electrons (Fig. 26.5) At the time of beam of electrons when hit by the electrons of the
transmission of beam of incident electrons atom of the object, the atom changes in an excited
through the specimen, the incident electron may state. This is due to the ejection of electron from
the orbit of the atom. Later on, the atom comes to
the stable unexcited state that occurs by shifting
Non elastic the electron from the outer shell to fill up the
interaction vacancy of the ejected electron. The excess
energy is released in the form of Auger electrons,
cathodoluminescence and X-ray.
Electron
Elastic interaction
Electron
Specimen
positively
charged nucleus
attracts the electron
Fig. 26.4 Elastic and non-elastic interaction of electron. Fig. 26.5 Schematic diagram shows interaction between
In elastic scattering the nucleus of the atom deflects the the electrons and the object. When the beam of incident
pathway of the primary electron of the beam. In non- electrons hits the majority of the electrons, they are trans-
elastic scattering, the principle electron of the microscope mitted through the object, and some amount of electrons
column is repulsed by the electrons of the orbit of the is backscattered. Occasionally electrons from the object
atom come out as secondary electrons
258 26 Electron Microscopy: Principle, Components, Optics and Specimen Processing
1. The sample should be thin and electron trans- Volume of Fixative The volume of fixative
parent. The thickness of the sample varies should be 15 times more than the volume of the
from 30–50 nm, and the upper limit of thick- sample.
ness is 100 nm.
2. The sample should be mechanically robust so Duration The average time of fixation is 9 h by
that it can withstand the handling in high 4% glutaraldehyde at room temperature and 1 h
temperature. for osmium tetroxide. The tissue should not be in
fixative for more than 12 h. Prolonged fixation is
The steps of sample preparation for TEM not recommended as this may extract the protein-
include [3]: aceous material from the tissue, and the proper
sectioning will be difficult. Under fixation may
1. Sample collection cause swelling of the mitochondria and disrup-
2. Sample fixation tion of the other cell organelles.
26.3 Sample Preparation for TEM 259
Moreover methacrylate may undergo sublima- • Diamond knives: The diamond knives are rel-
tion and disintegrate in the presence of high- atively expensive. The section quality of dia-
energy electron beam. mond knives is far better than glass knives.
These knives are more durable than the glass
Araldite: Araldite is an aromatic amine. This knives.
is one of the epoxy resins used for EM. Araldite
is used in combination with a hardener, an amine Semi-thin Sections It is the preliminary screen-
accelerator and a plasticizer. The amine accelera- ing procedure to see the adequacy of the sample.
tor accelerates the reaction between the resins. At first the resin-embedded blocks are trimmed
The components should be mixed properly to to expose the underlying tissue. Approximately
avoid the formation of any air bubbles. 1 μ thick multiple sections are cut from each
Epon: Epon is an alternative embedding block. The sections are picked up from the water
medium for EM. This is an aliphatic resin and has trough placed directly below the glass knife. The
low viscosity. Therefore it can infiltrate within semi-thin sections are dried in a hot plate at
the tissue more quickly compared to Araldite. 60 °C. The dried sections stick to the glass slide.
The semi-thin sections are stained with 1% tolu-
Polyester Resin Polyester resins have similar idine blue in 1% sodium tetraborate solution for
properties as that of epoxy resin. They do not 1 min to see the adequacy of the sample. If the
cause any cell shrinkage and polymerize uni- section contains the representative areas, then
formly. Vestapol W is the commonly used poly- further ultrathin sections are made from the
ester resin. block.
Ultra microtome
Mix lead nitrate and sodium citrate in distilled
water and shake them for 1 min.
Now add 8 ml 1M NaOH and mix them well.
Gently add 50 ml distilled water to dissolve the
precipitated lead nitrate. Keep pH 12. The solu-
tion will be stable for 6 months.
Fig. 27.1 Technical
requirements for quality Laboratory design:
control in the laboratory Space, ventilation, safety
are highlighted in this
diagram Scope and overall
facilities: Tests
Work definition:
Responsibility, license
Technical
requirements
for the quality Financial resources:
control Budget allocation
Equipment and
reagents: Good quality
Essential Technical Requirements for Quality etc. This knowledge of the financial budget
Control The essential technical requirements for gives the idea of the capability of the labora-
the quality control measures include (Fig. 27.1): tory to fulfil the customer’s need.
5. Laboratory equipments and reagents: The
1. Laboratory design: The laboratory should be standard equipments and reagents are needed
designed in such a way that there remains to provide good quality well-stained sections
enough space for receiving the sample, pro- and smears. The microtome, processing
cessing and staining and for the interpretation machine, etc. should be regularly updated.
area, storage, etc. There should be proper ven- There should be a proper log book mentioning
tilation and safety arrangement in the the use of the equipments, purchase date and
laboratory. expiry date of the chemicals.
2. Scope and overall facilities in the laboratory: 6 . Ensuring the quality of the processing and
Overall laboratory facilities should be clearly reporting: The quality of the processing
documented. Detailed description of all the should be regularly checked and recorded.
tests in the laboratory should be mentioned to Similarly the reporting quality should be veri-
the patients. fied periodically.
3. The work definition of the laboratory person- 7. Laboratory information service (LIS): LIS
nel: The work responsibilities of the different generates unique accession number of the
categories of the laboratory staffs should be specimen. This number provides the identifi-
clearly described. The staffs should be highly cation of the sample or section. The patient’s
competent with professional licence to prac- clinical history and other necessary
tise the respective work. information are listed in LIS. The final
4. Financial resources: It is necessary to know report is also entered in LIS, and the report
the overall financial budget allocation for lab- is recoverable instantly by the end service
oratory personnel, equipments, chemicals, providers.
27.2 Quality Control 265
Staining
Safety precautions
Microscopic examination
Analytical phase
Synoptic reporting
Fig. 27.2 Three important steps of quality control: pre-analytic, analytic and post-analytic phases
266 27 Quality Control and Laboratory Organization
27.3 External Quality Assurance • The floor and wall should be made in such
a way that they can be cleaned easily by
External quality assurance consists of: disinfectant.
• The room should have water supply, proper
• Proficiency test racks and closed almirah to keep the haz-
• Continuing medical education ardous chemicals separately. The process-
ing room must have a safety cabinet.
Proficiency Test The proficiency test is a volun- • The screening room should be isolated,
tary program. The various laboratories should take spacious and free from any noise.
part in the proficiency test to improve the diagnos- • The rooms for the secretarial staffs should
tic skill. In UK, the proficiency test is mandatory have adequate space for the typing equip-
for the reporting consultants who work in the NHS ment and furniture.
Breast Screening Program. Overall the proficiency 3 . Safety arrangement: The room should be
test is educational, and it points out the strength equipped with proper safety arrangement such
and weakness of the pathologists [7]. as fire extinguishers, etc.
The laboratory access pathways should be as fol- Qualifications and Training The technical
lows: sample collection, sample processing and staffs and the pathologists should have proper
staining and reporting, followed by the post- qualification and licence. Periodic evaluation of
examination area. the staffs should be done.
Each laboratory should have overall safety pre- • During preparing the diluted solution, the
cautions. The laboratory safety officers should concentrated acid or alkali should be
look after this following issue: added in water.
Overall security: This involves the general . Infective: The laboratory personnel should
D
security of the laboratory such as safety of the always take universal precautionary measures
equipment and reagents and prevention of entry because we do not know the HIV status of a
of any unwanted persons. sample [1, 2].
• Universal precautions (Box 28.1): Health
A. Security: education of the technical staff regarding
• Proper security of the laboratory staff, the universal precaution is very important
chemicals and valuable equipment is to prevent the transmission of infection.
mandatory. Universal precautionary measures imply
• Entry of unauthorized person should be that all the patients should be treated as a
restricted to the laboratory. potential source of blood-borne
B. Fire hazards: infections.
• The fire extinguishers, smoke alarm and • What is it? Universal precautions indicate
fire blankets are necessary in every to take adequate measures to prevent con-
laboratory. tact with various body fluids of the
• The laboratory personnel should know the patients. Various barrier measures are
basic operation protocol of the fire taken to avoid contact with body fluids
equipment. that are the potential sources of transmis-
C. Chemical hazards: sion of infection.
• The toxic and inflammable chemicals • The high-risk pathogens: The pathogens
should be in closed door fireproof metal that cause serious health hazards are hepa-
cabinet with original labels. titis B, hepatitis C and HIV.
• Never do suction by mouth. • Body fluids that need universal precau-
• Always put the alkali or acid in water dur- tions: This includes blood, peritoneal and
ing the procedure of dilatation. pleural fluid, CSF, semen, vaginal secre-
• Facilities to wash eye and shower in case tions, synovial effusion and faecal
of toxic exposure. material.
• Wear gloves, mask and laboratory coat • Body fluids that do not need universal pre-
during dealing with chemicals. cautions: These are faecal material, urine,
• Hand-washing: Simple maintenance of hand • Infective agents with serious health haz-
hygiene is the single most important factor to ards: hepatitis B, hepatitis C and HIV
prevent the transmission of infection. The • Body fluids that need universal precau-
cleaning of hands by anti-infectant soap tions: blood, peritoneal and pleural
removes the bacteria. The approved alcohol- fluid, CSF, semen, vaginal secretions,
based products such as gel or foam are supe- synovial effusion and faecal material
rior as these substances have better • No need for universal precautions: fae-
microbicidal activity and do not produce dry- cal material, urine, nasal secretions,
ing effect. Moreover no water is needed to sweat, vomitus and sputum unless con-
clean with alcohol-based cleaning agent. taminated with blood
• Gloves: Gloves prevent the blood or contami- • Measure:
nated substances to have direct contact with –– Hand-wash
the skin. Latex or nitrile gloves are better than –– Gloves
vinyl gloves. The gloves should not be washed –– Mask
for reuse as the removal of the micro- –– Goggles
organisms is not always possible from the –– Apron
gloves. –– Take caution with sharp objects
• Isolation gown: The isolation gown helps to –– Proper discarding of the contami-
prevent contamination with blood or mucus nated waste
product with the skin. It should only be worn –– Cleaning the area
when there is a chance of contamination of
blood or blood products. For routine labora-
tory work, wearing of simple laboratory gown
is enough. agents. Mask prevents transmission of infec-
• Mask: The mucus membranes of the upper tion from the patient to the healthcare person-
respiratory tract are vulnerable to infecting nel or vice versa. The masks may be of variable
sizes with different filtration capacities. The
Hand types of the mask depend on the need of the
Wear Wear
wash
gloves
particular staff.
masks
• Goggles: The various viral infections and
Staphylococcus aureus can be transmitted by
direct contact of splashed blood or touching
Universal
the eye with the contaminated hand to the eye
precautions
mucosa. Goggles should be used to prevent
the transmission of infection through the eye
mucosa.
Take care • Precautions from the sharp objects [3, 4] (Box
Wear Proper
of
aprons
sharp disposal 28.2): The most important pathogens that can
objects of waste be transmitted through needle prick injury are
HIV, HBV and HCV. The injury may happen
Fig. 28.1 Schematic diagram of universal precautionary during (1) recapping the needle, (2) transfer of
measures in laboratory the blood from container to container and (3)
28.1 Laboratory Waste Disposal 273
improper disposal of the needle. Following Basic Norms to Dispose the Waste Material The
precautions are helpful in needle stick injury: basic norms to dispose the waste material are:
–– Do not bend or recap the needle.
–– Use needle cutter to cut the needle. • Do not store waste in metal container.
–– Put all the sharp objects in proper • Do not store the chemical waste under the
container. fume hood.
–– Take universal precautionary measures. • Properly label the containers of the waste.
–– Written document on post-exposure • Store waste only in a closed container which
situation. is leakproof and no chance of puncture in case
–– Post-exposure follow-up and evaluation. of sharp material.
274 28 Laboratory Safety and Laboratory Waste Disposal
• Use only one type of container for the particu- hypochlorite generates chlorine, so it is
lar type of waste. highly corrosive and should not be kept in
• Do not keep incompatible chemicals in a same a metallic container.
container such as acid and alkali should not be B. Iodophors: Iodophores are the disinfectant
kept together. containing iodine in aqueous solution.
Betadine and povidone-iodine are widely
Table 28.1 highlights the different types of available commercially.
waste and their treatment protocol. C. Quaternary Ammonium Compounds: These
are also good detergents and disinfectants.
These compounds act against various bacteria
28.2 Disinfectant Used and viruses. However their action diminishes
in for the Contaminants with organic matter and many detergents.
These compounds are good for cleaning the
A. Chlorine-Based Compounds: laboratory floor.
• Sodium hypochlorite (NaOCl): It is a D. Phenolics: These are derivatives of phenol and
rapid oxidant material and is a broad spec- act by damaging the membrane of the bacteria
trum disinfectant. Chlorine is generated and fungi. Lysol is the widely available com-
from the diluted mixture and works as dis- mercial product. Many phenolic compounds
infectant. For laboratory purpose 10% are inactivated by hard water, and so it is pref-
sodium hypochlorite solution is used as erable to dilute them with distilled water.
chemical disinfectant. The solution should E. Others: Acid, alkali and alcohols are also
be made fresh every day. As the sodium used as disinfectants.
References 275