ROLE OF

INTRAOPERATIVE
CYTOLOGY IN TUMOR
DIAGNOSIS

Presenter: DR RITURAJ
Moderator: DR SWATI SINGLA
Chairperson: DR MEETU AGRAWAL
 OVERVIEW
HISTORY

INTRODUCTION to Intraoperative consultation

PREREQUISITES for Intraoperative consultation

GOALS of Intraoperative consultation

ADVANTAGES of intraoperative cytology (IOC)

LIMITATIONS of IOC

TECHNIQUE: Methods, Fixation and Staining

APPLICATIONS of IOC
 HISTORY

• 1891- William S. Halsted requested intraoperative consultation on a

mastectomy specimen- William H. Welch performed frozen section,
but procedure required an hour & results were not available until
after operation was completed.

• 1905- Louis B. Wilson published a frozen section technique that could

be performed in a few minutes
• 1927 - Leonard S. Dudgeon and Vincent Patrick at the University of
London used rapid cytological diagnosis with reliable accuracy rates.

• After these initial trials, the use of cytology smears during

intraoperative consultation has often been neglected in favor of
traditional examination of frozen sections.
 “When cancer becomes a
microscopic disease, there must be
tissue diagnosis in the operating
room.”
-Joseph Colt Bloodgood, 1927
 INRODUCTION

• INTRAOPERATIVE CONSULTATION is an important part of pathology,

where the goal of pathologist is to guide the surgeon to make
clinically relevant intraoperative decisions.

• Close cooperation between surgical and pathology teams is

important to make sure the patient receives the optimal treatment.
 Intraoperative
consultation

Frozen Intraoperative
section cytology
 PREREQUISITES
• Requisition form: Patient identification, surgeon name, operating room
number (including phone number) are all essential information

• Age and Gender

• Prior history of malignancy/surgery/medical treatment

Metastasis must always be considered

Treatment-related changes can be mistaken for malignancy

• Type of specimen submitted

Location

Biopsy or complete excision

Orientation

• Imaging findings
Essential to establish differential diagnosis
May be necessary to find lesion in large resections
• If purpose of IOC is not clear, pathologist should discuss with surgeon

• Communication with surgeon is very important

• All this information should be recorded on requisition form and

available to pathologist reviewing case for final sign-out.
 Goals of Intra-op consultation
1. Gross and/or microscopic diagnosis to guide intra or perioperative
patient management.

2. Confirm presence of lesional tissue for diagnosis on permanent

sections.

3. Adequacy of the tissue for ancillary studies to be used for

diagnosis, treatment, or research . (Lymphomas, Sarcomas,
Pediatric tumors)
 ADVANTAGES
• Accurate and fast method (whole process takes about 10-15 min)

• Cost efficient, doesn’t require special equipment.

• Better preservation of nuclear and cytoplasmic details

• Complete sampling is possible for a large or multiple specimens and

specimens with variegated areas and also in case of sentinel lymph
node assessment
• Tissue architecture is preserved for permanent sectioning

• Prevents contamination of cryostat (in case of infected sample)

• Helps academic purpose

• Shorter learning curve for pathologist.

• Freezing artifact is not introduced into the tissue.

 LIMITATIONS

• Margins and architecture of tissue can not be assessed (Difficulty in

differentiating invasive carcinoma from in-situ counterpart)

• The depth of infiltration cannot be analyzed with intraoperative

cytology.

• Scirrhous lesions very difficult to assess

 SITUATIONS WHERE FROZEN
SECTION IS MANDATORY
• MARGINS

• STROMAL INVASION

• DEPTH OF INVASION

• GROSS/CYTOLOGIC DISCREPANCY

• INADEQUATE SMEAR

• SPECIFIC REQUEST
TECHNIQUE
(Methods)
imprinting
squashing

IOC
(METHODS)

scraping
spreading
• The choice of the method depends on the size and consistency of the
tissue.

• Hence, gross examination of the specimen is an important aspect of

IOC.

• It is important that excess fluid (blood and saline) is gently blotted

before proceeding to minimize the effect of obscuring red blood cells
and artefactual shrinking or blowing up of the cells.
IMPRINT

• Freshly cut surface of the specimen

is gently pressed onto a glass slide.

• Misrepresentation of the cell’s

shape can be prevented by
restricting a gliding movement.
• This method tends to provide less cellular harvest but causes the
least cytological distortion.

• Imprinting is the method of choice in high-grade tumors such as

lymphomas and small cell carcinoma, where the tumor cells are most
fragile.
SCRAPING

Representative tissue taken from the specimen using edge of slide or

blunt edge of surgical blade
Obtained material is smeared on glass slide
SQUASH PREPARATIONS

Tissue is crushed between two slides to flatten out its matrix

Slides are pulled in opposite direction and both are used for staining
MINCING (mash and move)

• When the tissue is firm and unyielding, one can mince a small piece
on the glass slide using the scalpel blade.

• The tissue fragments are then dragged along the slide to obtain a
harvest of cells.
SPREADING

Mucinous tumors can be spread out in circular

motion to form an even layer
INTRA OP FNAC

• Material is aspirated with or without suction using needle and

syringe.

• Aspirated material is smeared on a glass slide

• It has limited and controversial role as a tool for intraoperative

cytology

• Requires trained staff in the operating room

Stains and Fixatives
• These smears can be stained with either Toluidine blue, Diff-Quik, PAP
or hematoxylin and eosin (H&E) .

• PAP and H&E stained preparations require immediate fixation in

95% alcohol, whereas TB & Diff-Quik stained preparations must be
air dried before staining.

• Different parts of the specimen can be applied to the same slide in

case of air dried smears.
FIXATION

Required to:

• To improve the staining potential of cell parts.

• To change the cytoplasm from an aqueous phase to a solid phase.

• To render the proteins insoluble (by denaturing them).

• To make the cells resistant to shrinkage and distortion.

WET FIXATIVES
COATING FIXATIVES
Combination of alcohol and a waxy base

• CARBOWAX- coats and protects the cells; keeps the cells from drying
out
• The coating has to be removed before staining by soaking the slides in
95% ethanol.

• SPRAYS- most common method; there are aerosol and non-aerosol

sprays. Spray Site, Cytospray, Cytofix, etc. (distance 15-25cm)
TOLUIDINE BLUE

• Discovered by William H Perkin in 1856, used as a dye since then

• TB is basic, metachromatic dye with high affinity for acidic tissue

components.

• Majority of the dyes stain tissues in differing degrees of intensity of the

same color, however, certain tissue components, which in the
presence of certain basic dyes stain a color other than that of the dye.
• The tissue is said to exhibit metachromasia and the dye as a
metachromatic dye.

• The dyes exhibiting metachromatic properties are mainly of thiazine

group (TB, azure A, azure B, methyl violet, safranin, and acridine
orange)
Staining procedure

• Two to three drops of toluidine blue on air dried smear.

• Keep for 10-15 sec

• Excess stain should be removed.

• Wet mount

• The slide is ready for a preliminary evaluation in about 1 minute.

DIFF QUICK STAIN
The Diff-Quik stain consists of 3 solutions:
• Modified Wright-Giemsa/Romanowsky stain used primarily for air-dried
cytologic preparations (Bernard Witlin in 1970)
1. Diff-Quik fixative reagent
2. Diff-Quik solution I (eosinophilic)
• Especially helpful in evaluation of cytologic details
• Xanthene dye
• Requires monolayers of cells as stain does
• pH not penetrate well
buffer
3. Diff-Quik
• Hence, touch preparations may be more solution IIthan
appropriate (basophilic)
smears
• Thiazine dye
• Requires only 15-30 seconds depending
• on thickness of preparation
• pH buffer
pH buffer
Staining procedure

• Allow slides to air dry

• Fix in 95% ethanol - 10 dips

• Place in solution 1 - 12 dips

• Place in solution 2 - 6-8 dips

• Rinse with distilled water - 20 dips

• Mount and analyse

PAP STAIN

• Developed by Dr. G Papanicoulou in 1943. (Father of cytology)

• The classic form of Pap stain involves five dyes in three solutions.

• Standard PAP staining requires about 30 minutes

• RAPID PAP staining requires only 4-5 minutes

• Multiple variations of RAPID-PAP are present

Staining procedure:
1. Fixation with ethanol.
2. Hydration – 10 passes under running water.
3. Nuclear stain – 45 sec. Wash under tap water
4. Scotts water- 10 sec. Wash under tap water
5. Dehydration – 30 sec.
6. Working Cytostain- 15 sec. Wash under tap water
7. Dehydration – 30 sec. Dry.
8. Xylene
9. Mount with DPX
Ultra fast PAP- 90 seconds
• Background is better in UF-PAP stain than in the standard PAP stain.

• The rehydration of air-dried smears in saline causes lysis of the

RBCs- hence a better interpretation is possible.

• UF-PAP procedure uses less alcohol and xylene changes with the
omission of O-G-6 components

• Should be careful in cases of SCC

APPLICATION IN VARIOUS
TUMORS
 CNS
• 1930: Eisenhardt and Cushing advocated the use of touch preparations for rapid
diagnosis of brain tumors.

• Since then Cytology of the CNS has become of capital importance in neuropathology.

• Stereotactic CT guided biopsies provide with tiny biopsy sample from brain tissue,
hence, Intraoperative consultation on a brain lesion is mostly done by a smear or a
crush preparation of the submitted tissue sample (often may be too small or fragile
for a frozen section.)
Smear preparation

Touch preparations:

• Best for lymphomas and pituitary adenomas

Scrape preparations:

• Best for metastatic carcinoma and meningioma

Squash preparations:

• Glial tumors: Neural cells have long, inter-tangling, processes that may not be well
represented in touch preparations or smears

• Meningiomas and schwannomas: May be too fibrous to squash well

• In non-cohesive processes, cells grow without significant
intercellular attachments - pituitary adenoma

• Cohesive smears -schwannoma

• Intermediate smears – epithelial smears (metastatic tumors)

• Glial tissue smears - Reactive gliosis

Necrotic debris smear-This fragment of tissue from a glioblastoma is necrotic.

The pale, dead nuclei (arrows) are diagnostic evidence of necrosis

PAY ATTENTION TO:

1. Relationship of blood vessels to neoplastic cells:

• Perivascular gradient pattern-gliomas especially astrocytomas

• Angio-centric and diffuse pattern-lymphomas and

• Randomized clusters with or without vascular affinity-metastatic

tumors
2. Blood vessel type: (Important for
grading)

• Low-grade gliomas and pituitary

adenoma display thin walled vessels.

• Glioblastoma and metastatic deposits

show thick vessels with endothelial
proliferation.
3. Type of background:

- Normal brain matter is characterized by a felt like background.

• Astrocytomas: usually demonstrate the presence of fine, well defined

glial processes in the background (fibrillary background)

• Oligodendrogliomas may have more of a pool table felt background

• Metastatic carcinoma and Lymphoma: fibrillary background generally

absent; often a felt pattern
Astrocytic Neoplasms-
Low grade astrocytoma (pilocytic) (grade 1)
• Relatively sparse population of uniform spindly cells with small clear
nuclei, fairly abundant clear cytoplasm with long thin like hair like
cytoplasmic processes.
• Rosenthal fibres may be present.
High grade astrocytoma (grade II,III)
• Higher cellularity, nuclei of irregular outline hyperchromatic but
fundamental structure similar to low grade astrocytomas.
Pilocytic astrocytoma- showing astrocytes with fibrillary processes and
Rosenthal fibers
Glioblastoma: Grade 4
• Radiographically shows low density area with central enhancing area of
necrosis and surrounding edema ( may be confused with abscess
clinically).
• Grossly may appear circumscribed intraoperatively but always infiltrative
microscopically.
 Smear cytology-
• structure of classic astrocytoma is lost.
• Many bizarre tumor cells of various sizes , mitoses and tumor giant cells
along with necrosis.
Glioblastoma- smear showing increased cellularity with pleomorphism
 Oligodendroglioma
• Derived from small glial cells with
short cytoplasmic processes.
• Smears show sheets of monotonous
small cells with scant clear
cytoplasm with halo like effect and
felt like background.
• Tumour cells have stippled
chromatin and scant pale cytoplasm.
 Ependymoma-
• Smear shows papillary groups around
blood vessels forming pseudo-rossettes
or seen as palisading tumor cells
arranged on both sides of long axis of a
vessel.
• Prominent fibrillary matrix is present
adjacent to blood vessels or at edges of
groups of tumor cells.
• The cells are oval to round with pale
stippled chromatin and one or more
nucleoli.
 Medulloblastoma

A. Cells with scant-to-no cytoplasm having nuclei displaying salt-and-pepper chromatin but
no nucleolus. Nuclear moulding reflects cellular cohesion in the presence of minimal
cytoplasm.
B. Occasionally forming pseudo-rossettes.
 Meningioma
• These are rubbery, firm and well demarcated tumours that have dural
attachment.
• Smears show cohesive clusters of meningothelial cells having eccentrical
nuclei and abundant tissue paper or delicate wispy cytoplasm.
• Whorl formation is variable but usually seen.
• Nuclear pseudoinclusions +/-
• Psammoma bodes can also be seen: clue to their presence on squash
preparation is grinding sensation during squash.
whorl or pearl like arrangement of meningothelial
cells and psammoma bodies
 Pituitary adenomas
• The majority of the pituitary adenomas have
soft texture .
• Smear easily into thin film.
• Monotonous proliferation of round to oval
cells in acinar pattern
• In typical cases, the nuclei are centrally
placed, very uniform and rounded in shape,
and they have finely speckled chromatin with
no distinct nucleolus.
Primary CNS lymphomas

• Smear shows monomorphic population of cells which show a wide range

of nuclear pleomorphism.
• Most cells have distinct cell borders but scant cytoplasm, high
nucleus/cytoplasmic ratio, irregular nuclear contours, granular chromatin
and prominent nucleoli.
  Schwannomas
• Grossly the tumor has a firm rubbery texture with a whorled appearance; cystic
• Degeneration and hemorrhage may be present.

Squash preparation of schwannoma- showing cohesive cells with frayed rope appearance.
 Metastatic neoplasms

• Cellular cohesiveness is a striking feature in epithelial neoplasms

• Moulding of adjacent cells in the clusters is a helpful feature

• The degree of pleomorphism and the cytological features vary in the

many types of tumors.

• Glandular differentiation and mucin production may be seen in

adenocarcinomas.
PITFALLS:

Presence of Macrophages : Strong indicator of non-neoplastic diagnosis

• Demyelination, infection, infarct - Reconsider diagnosis of tumor

• However, may be seen high-grade tumors on treatment

Necrosis Without Cellular Component: Specimen inadequate

• Insist on additional material to diagnose lymphoma, metastasis, glioma, infarct,

or infection
Necrosis Following Radiotherapy

• Usually accompanied by other evidence, e.g., vascular wall fibrinoid

change or hyalinization, endothelial atypia

• In treated low-grade gliomas, necrosis may not indicate anaplastic

progression

• Report as “glioma with necrosis and radiation changes, grading

deferred”, unless obvious high-grade features (pleomorphism,
vascular proliferation, mitotic activity) present
Presence of Rosenthal Fibers

• Seen in slow-growing or longstanding lesions, including non-

neoplastic

• By itself, does not imply pilocytic astrocytoma

• But when not seen in small biopsies, may make diagnosis of pilocytic
astrocytoma difficult

• Report as “astrocytoma with piloid features”

Uses
• Rapid, tissue-sparing diagnosis of metastatic disease (carcinoma,
melanoma), pituitary adenoma, craniopharyngioma

• Aid adequacy assessment in stereotactic core needle biopsies

• Differentiate between : Neoplastic vs non-neoplastic, Type of

neoplasm (glial vs non-glial) and Grading of neoplasm

• Adjunct to frozen section in diagnosis of primary central nervous

system lesions
 OVARIAN TUMORS

• The scrape technique is the best method for preparing the smears.

• Cellularity is highest in scrape smears compared to other techniques

and morphological preservation is also better. 

• Role of intraoperative and preoperative FNAC is controversial as it

may lead to seeding of tumor in peritoneal cavity.
Serous tumors

• All the benign serous tumors are predominantly cystic filled with pale
straw-colored fluid.

• Smears show papillaroid clusters as well as monomorphic sheets of

epithelial cells with small dark, bland nuclei, and moderate to
abundant cytoplasm.

• The background is usually clean in majority of cases.

 Borderline serous tumors

Branching papillary pattern with peripheral

detachment of small epithelial nests. Low nuclear
atypia. Clean background.

High cellularity. 2D and 3D dense cell groups.

Scarce single cells. Low to moderate nuclear
atypia. Oval and round nuclei, small, fine nucleoli,
scant cytoplasm.
 Serous cystadenocarcinoma

• Numerous single cells and small

3D groups.
• Loss of polarity and
cohesiveness.
• High nuclear atypia. Irregular
chromatin and prominent or
macro nuclei.
Scrape smear moderate pleomorphism, inflammatory
background and mitosis (inset).
Mucinous tumors

Columnar cells forming honeycomb-like clusters with basally placed

nuclei and empty looking cytoplasm in a mucinous background 
borderline mucinous tumor

• Cohesive sheets of mucin-secreting

columnar cells with moderate
degree of cell overlapping and many
dispersed cells.

• There is mild to moderate degree of

nuclear atypia and pleomorphism 
Mucinous adenocarcinoma

• Mostly solid cystic.

• The smears show dirty mucinous

background and discohesive
clusters as well as dispersed
moderately pleomorphic cells.

• Few of the cells also show mucin

vacuoles 
 Solid Epithelial Tumor: Brenners

The smears are cellular showing mild nuclear pleomorphism and prominent nucleoli
Presence of nuclear grooves highly suggestive of granulosa cell tumor (inset)
Germ cell tumors

Dysgerminoma

• Sheets of large poorly cohesive tumor cells having abundant clear cytoplasm with pale
nuclei and single nucleoli with lymphocytes against characteristic tigroid background

Embryonal and yolk sac tumor

• Small cells having vacuolated cytoplasm and prominent nucleoli arranged singly or in
papillary clusters

• Presence of hyaline globules indicate yolk sac tumor, Schiller-Duval bodies may also be
seen
Teratoma

• Characteristic gross appearance

• Amorphous material (sebum) mixed with squamous epithelial cells and inflammatory

cells.

• Presence of hairs and columnar epithelial cells of respiratory or intestinal type are

indicative of benign teratoma

• Presence of undifferentiated small malignant cells resembling neuroblastoma indicate

malignant teratoma
 SENTINAL LYMPH NODE (SNL)

Sauer et al (2003) reported that imprint

• Node should be serially sectioned at < 0.2 cm intervals and all surfaces carefully examined

• Scrape all
cytology ofcutsentinel
surfaces to
lymph make
nodes is cell-rich
rapid andsmear(s)
or If multiple nodes are sampled, use separate blades and make separate smear(s) for
reliable and provides same-day diagnosis for
each node (imprint method can also be used)

• Focus scrape preparation on gross target lesion to obtain higher concentration of lesional
counselling purposes and for planning future
cells
surgery.
• If lesion is cytologically indeterminate, prepare frozen section to assess
 PARATHYROID EXPLORATION
• Pathologic confirmation of 4 glands microscopically

• Intra op cytology distinguishes between-

Parathyroid

Thyroid

Thymus

Lymph node

Fat
Cytology :
• Smears are cellular - loose cluster of cells with delicate ill defined cytoplasm
variably sized round nuclei

• Intracytoplasmic vacuoles indicate parathyroid adenoma or hyperplasia

• LN: lymphoglandular bodies

• Thymus: Hassals corpuscles

• Thyroid: Follicles and colloid

• Adipose tissue: Hypocellular with fat fragments

 THYROID

• Intraoperative diagnosis of thyroid nodules is of significant assistance

to the surgeon who intends to perform a procedure more extensive
than local excision or lobectomy for primary carcinoma of the thyroid
gland.

• Also applicable in cases where preoperative FNA can not be done or

it is inconclusive.
 BREAST
• The importance of IOC in breast tumors have reduced because of newer
surrogate molecular classification and better preoperative assessment
of patients.
 ASSESSMENT OF MARGINS

• While operating a malignant tumour it is critical to obtain clear margins to

minimize local recurrence.

• However, avoiding multiple re-excisions for margin clearance helps optimize

cosmetic results in patients undergoing breast conservation surgery.

• Available literature suggests Intra-operative touch preparation cytology may

decrease the need for multiple re-excisions and thereby improve cosmesis.
THANK YOU!!!!!!!