GROSS TECHNIQUES IN

SURGICAL PATHOLOGY
Introduction

The routine work associated with a

surgical pathology specimen includes
gross & microscopic examinations.
Gross examinations give an idea about
size ,shape of specimen &any gross
abnormality like ulceration,
nodularity.
The dissection, gross description
&selection of sections for
microscopic study is a crucial part of
pathologic examinations
Preparations of sections for histopathology

1- paraffin embedding method ( the

routine & widely used procedure).
2- frozen section (intra-operative).
3- cytological diagnosis (exfoliative &
fine needle aspiration cytology.
4- Electron microscopy and
Immunohistochemistry
Paraffin embedding method

Include the following principal steps:

1- fixation:- to preserve the
tissue, fixatives include
formaldehyde, Zenker’s solution,
picric acid, Bouin’s solution,
The best fixative is 10% buffered
formalin
Purposes of fixation

 Prevent autolysis and purification.

 Preserve tissue details as nearly as
possible to living state.
 Hardening the tissue by coagulating
proteins.
 Renders the tissue receptive to
subsequent staining (by altering
refractive index of various cell
components).
 Prevent damage during subsequent
procedure.
Formalin

 10% formalin in neutral buffer (10

volumes formalin, 90 volumes NS)
 The speed of fixation of the most
used fixative 10% buffered formalin
is about 1 mm/hr.
 The volume of the fixative should
be at least 10 times that of the
tissue.
 Fixation can be carried out at( room
temperature) or in the case of
large specimen at 4°C .
 The freezing point of 10% formalin
solution is (-3°C).
 N.B. Pure formalin is a concentrated
40% solution of the gas
formaldehyde in water.
Advantages of Formalin

1- cheap. always available.

2-good penetration into tissue.
3- cause little shrinkage.
4- preserve RBCs & fatty tissue.
5- special stains can be used on
tissues fixed with it.
6- preserve color of the tissue.
7- good hardening.
8-Suitable for making frozen sections.
Disadvantages of Formalin

1- if tissue preserved in formalin for long

time, formic acid will be formed which
affect stainability of tissue with different
stains, so it should be changed every 3-6
months.
2- when formalin solution is stored for long
period a white precipitate of para
formaldehyde which will not affect the
efficiency of formalin as a fixative & can
be removed by alcohol.
3- Color reaction with amyloid is less.
Sampling for histological examination:

 After fixation with 10%

formalin, wash with
water to remove the
fixative.

 Dissection
 Trimming and
orientation in tissue
cassette
 specimens are cut in
pieces measuring 10-15
mm on the sides and 2-3
mm in thickness.
2- Dehydration:- get rid of water to
facilitate penetration and infiltration of the
tissue with paraffin. Progressive ascending
grades of alcohol (70, 80, and 90,100%) to
prevent shrinkage of tissue
 3- Clearing:- :xylol
 Paraffin wax is insoluble in alcohol, so
alcohol must be replaced by a fluid that is
miscible or solvent for paraffin wax.
 Makes the tissue translucent.
III. Dehydration
D. Procedure
1. automatic
tissue processor
a. overnight
2. Baths: water,
70,95,100,100 %
alcohol
3. Clearing agent:
2 baths of xylene
 4- Paraffin impregnation. in melting
paraffin
 To enable the natural cavities of tissues
to be filled with paraffin thus preserving
relationships to each other.
 To surround the tissue by a plastic substance
to support the tissue when making
slides.

5- Embedding:- to make the tissue as a

block of hard paraffin
4) Paraffin
impregnation A.
A.Replace xylene with
paraffin
B. Immerse in melted
paraffin
1. ~55o C MP
C. Remove all bubbles,
xylene
D. Procedure
1. Two baths of melted
paraffin
VI. Embedding
D.Procedure
1. Place tissue
cassette in melted
paraffin
2. Fill mold with
paraffin
3. Place tissue in
mold
4. Allow to cool
6-Sectioning:- by using a
microtome, the tissue is sliced into
very thin sections ranging from 4-6
micrometer in thickness.

7- Attaching sections to
the slides. Sections are
transferred to slides and kept in an
incubator for few hours.
VII. Sectioning

Rotary microtome
5-10 mm

1-1
VII. Sectioning
E. Procedure
1. Place tissue block in microtome with
wide edge of trapezoid lowest, and
parallel to knife
2. Advance blade toward block
3. Begin sectioning
VIII. Attaching
sections to the
A. 40 C water bath
o slides.
1. Flattens paraffin section
2. Permits mounting on slide
B. Gelatin & albumin
C. Glass slides
D. Oven / air dry
8- Deparaffinization:- by using xylol
& alcohol until the paraffin is dissolved.

9- Staining:- the standard staining

method is H & E which stain the nucleus
blue (basophilic) & the cytoplasm pink-red
(acidophilic).

10- Mounting:- by using DPX or

Canada balsam &cover slip.
IX. Staining
G. Procedure
 Xylol to dissolve the wax.
 Hydration
 staining
 Dehydration
 Xylol to remove alcohol
X. Coverslipping
A. Coverslip & mounting medium (not
miscible with water)
B. Dehydrate
C. Clearing agent
D. Permount
XI. Pitfalls
A. Poor fixation (poor structural details)
B. Inadequate dehydration
C. Contaminated xylene (milky)
D. Poor paraffin impregnation (bubbles,
poor support)
E. Embedding: orientation, bubbles
XI. Pitfalls
F. Poor sectioning
1. knife marks (scratches perpendicular to
knife edge)
2. compression (waves parallel to knife
edge)
XI. Pitfalls
G. Mounting sections
1. folds & tears
2. excess albumin (stain)
XI. Pitfalls
H. Staining
1. inadequate rehydration (uneven
staining)
2. too dark or too light (timing off)
3. inadequate agitation
XI. Pitfalls
I. Coverslipping
1. Bubbles
XI. Pitfalls
I. Coverslipping
2. excess Permount
3. two coverslips
XII. Interpretation

A. Artifacts
B. 3D from 2D

1-30
FROZEN SECTION
TECHNIQUE
 It is indispensable technique for
rapid histopathological diagnosis
and intraoperative surgical
consultation without use of:
 dehydrating solutions
 clearing agents or
 embedding media
Indications and Advantages:

1-Quick method for urgent surgical

diagnosis
2-Guides extent of operation of tumor -
local excision or radical excision.
3- For demonstration of fat
4-Identification of minute structures to be
removed, vagus nerve in vagotomy
Sympathetic ganglion, parathyroid gland,
etc....
5-Helpful in enzyme histochemistry,
immunohisto- chemistry, tumour marker
detection antigen and antibody
detection.
Disadvantages:

1-Requires sophisticated-cryostat.
2-Thick section:10 µm or more in
thickness
3-Structural details become
distorted
4-Staining is not satisfactory in
case of unfixed sections
5-Freezing artifacts are observed
6-Bone sectioning is problem, skin
can be difficult.
Freezing process is achieved by :

 Cryostat
 Freezing microtome
 Freez drying method
The Cryostat
 The cryostat is a refrigerator cabinet
containing a microtome. All the controls
are operated from outside (the
temperature, the knife, the block
movement etc.)
 No fixation of tissue is needed, fresh
unfixed tissue are used and freezed
directly.
 Sections are picked directly on the slide
(no need for water bath)
 The knife must be very sharp, and the
edge should be free from defects (a
wedge-shape knife is used)
 An anti-roll plate or device is used to
The freezing agents used are:-

 liquid nitrogen at -190 °C

 solid carbon dioxide at -70 °C
 carbon dioxide gas at -70 °C
 aerosol spray at -50 °C
 Occasionally fixation is needed;
the fixative used is formol
calcium.
Tissue preparation

1-Fresh tissue block of

approximately 5mm thick with
parallel sides
2-The tissue can be fixed with
10%formalin or formol-alcohol.
3-Tissue can boiled in a beaker
with the fixative for 30-60
seconds
4-Tissue is washed in distilled
water.
Staining of frozen section

Haematoxylin -eosin method

(H&E): rapid method.
Biopsy

 Incisional biopsy: only a portion

of the lesion is sampled so the
procedure is strictly diagnostic.
 Excisional biopsy: the entire lesion
is removed usually with a rim of
normal tissue so the procedure serves
both a diagnostic and therapeutic
 an excisional biopsy is usually done in
small lesions.
Incisional biopsy
Incisional biopsy
Excisional biopsy
Excisional biopsy
General rules for the biopsy procedure:

 The largest the lesion, the more

numerous the biopsies that should be
taken from it, the diagnostic area may
present focally.
 In ulcerated tumors, biopsy is taken
from the periphery including both
normal and diseased tissue.
 Biopsy should be deep enough that
the relation between tumor and
stroma can be properly assessed.
 Deeply seated lesions are sometimes
accompanied by a prominent peripheral tissue
reaction which may be characterized by chronic
inflammation, hyperemia, fibrosis and metaplastic
bone formation. If the biopsy is too peripheral, this
may be the only lesion obtained.
 When several fragments of tissue are obtained,
they should be studied microscopically.
 Crushing or squeezing the tissue with the forceps
should be carefully avoided.
 Once the biopsy is obtained, it should be placed
immediately in a container with adequate fixation.
Exfoliative cytology:

 Examination of cells which detach

from tissues.
 Its clinical value is in:
 Early detection and diagnosis of
cancer.
 cytogenetic
 The study of hormonal effect on
certain tissues.
Early detection and diagnosis
of cancer.
 Cancer cells are less cohesive than
normal cells
 The study of morphological details of
individual cells gives valuable
information in the diagnosis of cancer.
 This is very important in the diagnosis
of cervical carcinoma and lung cancer
using papnicolaou staining technique
(sputum, pleural fluid , ascitic fluid,
urine)
Insitu carcinoma, urinary
bladder
 Smears are obtained by direct
scraping from the cervix fixed
immediately ,then stained with pap.
 It is important to emphasize that
negative cytology results indicate
only that cancer cells were not
present in the slide.
Cytogenetic

 Demonstration of the nuclear (sex

chromatin) Bar body in epithelial
cells of buccal and vaginal smear are
helpful in studying sex anomalies
such as Klinefelter’s syndrome and
Turner’s syndrome.
The study of hormonal
effect on certain tissues.
 Examination of vaginal smears
stained by PAP stain can give
valuable information regarding the
hormonal state.
 Epithelial cells of the female genital
tract are under direct influence of
hormonal activity especially estrogen
and progesterone.
FNAC ( Fine Needle Aspiration Cytology)

 The examination of cells obtained by

aspiration through a fine needle in solid
organs or tissue masses.
 Materials :
 Syringe : disposable plastic syringe, 10-20
cc
 Needles: fine needle ranges from 25 to 18
gauge or 0-6 mm to 0-9 mm in outer
diameter. Lengths vary from 1cm to 20 cm
 Glass slides
 Evaluation in relation to
histopathology, age, sex, location,
character of the mass and clinical
impression.
Advantages:
 Less trauma
 Rapid, accurate, cost effective, little
complications, safe, gives a great
psychological improvement for the
patient relieving anxiety.
Liver biopsy
Disadvantages:

 Loss of tissue architecture

 the needle may miss the lesions.
 Can give false positive result in the hand of
inexperienced pathologist.
 Smears sent to the cytology laboratory from outside
are poorly prepared and therefore difficult to
evaluate.
 A negative result is not helpful if clinical evidence
suggest the presence of malignancy so here we
should do other investigation (biopsy).
 Minor complications: Bleeding tendencies spread of
infections, pneumothorax, syncopal attacks, and
dissemination of malignancy along the needle tract.
Electron microscopy

 Main applications of EM in diagnostic

pathology are in the field of the tumor
pathology and renal pathology.
 It is very useful in determining the
histogenesis or differentiation of various
tumors, but unfortunately has not
shown consistent differences between
reactive conditions, benign tumors and
malignant tumors of the same cell type.
 Lesions of controversial nature in which EM has
provided useful information and sometimes
settled the histogenesis are (e.g. granular cell
tumor, schwannoma, sarcomatoid carcinoma,
carcinoid tumors of various sites).
 The best chance for EM to be of utility (benefit)
is when the pathologist has already formulate a
definite differential diagnosis between two or
three entities at the light microscopic level and
examine the tissue by EM searching for specific
markers expected in each of those entities.
 Limitations of EM:
 Only a small sample of the tumor can
be studied.
 Rarity or paucity of truly specific
ultrastructural features.
 Possible misinterpretation of
entrapped non neoplastic elements
as belonging to the tumor.
The main diagnostic situations in which EM is of help:
 Differential diagnosis between melanoma, sarcoma and
carcinoma.
 Differential diagnosis of anterior mediastinal tumors
between thymoma, thymic carcioid , malignant lymphoma
, seminoma.
 Differential diagnosis between adenocarcinoma and
mesothelioma.
 Differential diagnosis of small round cell tumors, between
Ewing sarcoma, malignant lymphoma, embryonal
rhabdomyosarcoma.
 Differential diagnosis of spindle cell tumors of soft tissues.
 Differential diagnosis between endocrine and non-
endocrine tumors.
Immunohistochemistry

 Is the application of immunologic

principles and techniques to the
study of cells and tissues.
 Several procedures are available ,
the two most commonly used are:
 Peroxidase-antiperoxidase immune
complex technique.
 Biotin –Avidin immunoenzymatic
technique.
Using monoclonal antibodies directed against certain antigen epitopes.
 Actin: smooth muscle cell (contractile protein)
 αfetoprotein: glycoprotein, Germ cell tumors
 Desmin : is muscle type intermediate filament found in striated and
smooth muscle.
 EMA: excellent marker of normal and neoplastic epithelium.
 F VIII R Ag: synthesized by endothelial cells of blood vessels.
 GFAP( Glial fibrillary acidic protein ): cytoplasmic intermediate filaments
. It is present in normal and neoplastic astrocytes , natural and reactive
ependymal cells , developing and neoplastic oligodendrocytes.
 HCG: normally secreted by the syncitiotrophoblast , composed of α and
β subunits . Ab prepared against β subunits is employed to detect
trophoblastic differentiation in germ cell tumors.
 S100 protein: acidic dimeric calcium binding protein present in the
cytoplasm and nucleus of glial and Schwann cells , melanocytes,
chondrocytes , its main use is in the evaluation of tumors presumed to
be malignant melanoma.
Advantages :
 Sensitive and specific.
 Applied to routinely processed material
even if stored for a long time.
 Accurate correlation with traditional
morphological parameters.
 Compatible with most of the fixative
currently used.
 It can be used in conjunction with other
techniques such as staining in the same
sections.