“A COMPARATIVE STUDY ON SQUASH, FROZEN

SECTION AND HISTOPATHOLOGICAL EXAMINATION IN

THE DIAGNOSIS OF CENTRAL NERVOUS SYSTEM

LESIONS”

Dissertation submitted to

The Tamil Nadu Dr MGR Medical University

In partial fulfilment of the requirements for the award of the degree of

M.D.PATHOLOGY

BRANCH III

INSTITUTE OF PATHOLOGY

Madras Medical College And Rajiv Gandhi Government General

Hospital, Chennai-600 003

The Tamil Nadu Dr. M.G.R. Medical University

Chennai-600 032

MAY 2018

CERTIFICATE

This is to certify that this dissertation entitled

“A COMPARATIVE STUDY ON SQUASH, FROZEN SECTION AND

HISTOPATHOLOGICAL EXAMINATION IN THE DIAGNOSIS OF

CENTRAL NERVOUS SYSTEM LESIONS” is the original work of

Dr.S.HEMALATHA, in partial fulfilment of the requirement for

M.D., (Branch III) in Pathology examination of the Tamil Nadu Dr.M.G.R.

Medical University to be held in May 2018.

Prof. Dr. Bharathi Vidhya Jayanthi Prof. Dr. R. Narayana Babu

Director and Professor, DEAN,

Institute of Pathology, Madras Medical College &

Madras Medical College Rajiv Gandhi Government General Hospital,

Chennai – 600003 Chennai - 600003

DECLARATION

I, Dr. S.HEMALATHA, solemnly declare that the dissertation titled

“A COMPARATIVE STUDY ON SQUASH, FROZEN SECTION AND

HISTOPATHOLOGICAL EXAMINATION IN THE DIAGNOSIS OF

CENTRAL NERVOUS SYSTEM LESIONS” is the bonafide work done by

me at the Institute of pathology, Madras Medical College under the expert

guidance and supervision of Prof. Dr. Bharathi Vidhya Jayanthi M.D.,

Director & Professor of Pathology, Institute of pathology, Madras Medical

College. The dissertation is submitted to the Tamil Nadu Dr. M.G.R Medical

University towards partial fulfilment of requirement for the award of M.D.,

Degree (Branch III) in Pathology.

Place: Chennai

Date: Dr. S.HEMALATHA

ACKNOWLEDGEMENTS

This thesis owes its existence to the support, help, inspiration and

guidance of several people. I must thank first our Dean,

Prof.Dr.R.Narayana Babu for allowing me to proceed with this study and

having permitted me to utilize the clinical material and facilities of this

hospital.

My heartfelt gratitude to my guide Prof. Dr. Bharathi Vidhya Jayanthi

Director & Professor of the Institute of Pathology for her encouragement,

motivation, monitoring and guidance during my study period. A teacher with

zenith of knowledge and wisdom in Pathology, she showed me the correct path

to go. I am indebted to her for her constant probing to get the best out of this

study. I am indeed privileged to have her as my guide.

I place my sincere thanks before Prof.Dr.K.Rama, Professor of

Neuropathology for her support and guidance.

I am also grateful to Prof Dr.A.Thiruvalluvan, Prof & Head of

Department of Neurosurgery, Madras Medical College, who with his

enthusiasm and support helped me in completing this thesis.

I sincerely thank my professors Prof.Dr.R.Padmavathi,

Prof.Dr.GeethaDevadas,Prof.Dr.SudhaVenkatesh, Prof.Dr.Ramamoorthy,

Prof.Dr.M.P.Kanchana Prof.Dr.Rajavelu Indira, Prof.Dr.S.Pappathi,

Prof.Dr.Selvambigai whose encouragement and support was paramount in

doing this thesis.

My very special thanks to Dr.S. Dhanalakshmi, Assistant Profesor

of Pathology who was my co guide, who persuaded me in continuing this

title for thesis and helped me by providing moral as well as material help.

Without her this thesis would not have reached its completion. I would

like to thank all my assistant professors for their valuable help and

advice.

I would express my heartfelt thanks to my husband for his constant

support, guidance and patience throughout this study.

I also thank my family members, friends and fellow postgraduates in

the department who were always with me. My special thanks to

Dr.Porchelvan who provided the statistical analysis.

Thank you all!

Chennai

Date: S.HEMALATHA

PLAGIARISM CERTIFICATE

This is to certify that this dissertation work titled “A Comparative

Study On Squash, Frozen Section and Histopathological Examination in

the Diagnosis of Central Nervous System Lesions” of the candidate

Dr.S.Hemalatha with registration Number 201513001 for the award of M.D.

Pathology (Branch III) . I personally verified the urkund.com website for the

purpose of plagiarism Check. I found that the uploaded thesis file contains

from introduction to conclusion pages and result shows 2 percentage of

plagiarism in the dissertation.

Guide & Supervisor sign with Seal

INDEX

S. No TITLE PAGE NUMBER

1 INTRODUCTION 1

2. NEED FOR THE STUDY 3

3. AIM AND OBJECTIVES 4

4. REVIEW OF LITERATURE 5

5. MATERIALS AND METHODS 31

6. RESULTS 35

7. DISCUSSION 61

8. SUMMARY 74

9. CONCLUSION 75

BIBLIOGRAPHY

ANNEXURES

INTRODUCTION

1

INTRODUCTION

Brain is the most vital organ in our body. It integrates all the system

and is important for coordinated function of the body. CNS lesions impair this

important role of the brain. Intraoperative diagnosis of central nervous system

(CNS) lesions is of utmost importance for neurosurgeons to modify the

approach at the time of surgery and to decide on further plan of management

1.There are basically three methods to obtain intra operative diagnosis viz.

touch preparation, squash smear, frozen section2.

Frozen section is being used by pathologists for over 100 years 3. But

the inherent soft nature of the CNS renders poor quality frozen sections 4. In

this regard squash smear preparations have an advantage over frozen sections

and they are increasingly being used by the neuropathologists to help the

operating surgeons.

Epidemiology:

Space occupying lesions of the brain are most commonly tumours,

primary followed by metastatic and other lesions such as non-neoplastic cysts,

abscesses, Tuberculomas, fungal granulomas, syphilitic gumma, cysticercosis

and hydatid cysts.

Primary CNS neoplasms account for 2% of all cancers and 20% of

cancers in children under15 years of age and most are neuro epithelial in

origin2. Among the primary CNS neoplasms glioblastomas and anaplastic

astrocytoma account for 35%, astrocytoma 13%, other neuro epithelial

neoplasms 8%, meningioma 17%, PNETs 3% nerve sheath tumours 8%, and

others 16%.

2

The SEER registry reports that the incidence of primary CNS tumours is

between 2.2 and 8.3 per 100,000 people per year, based on race and gender (for

all races, the incidence is 7.7/100,000 men and 5.4/100,000 women and the

median age at diagnosis is 55, and an age-dependent bimodal distribution is

observed, with the incidence estimated at 3.1/100,000 up to age 4, 1.8/100,000

from age 15 to 24 years, and a peak of 18/100,000 around age 65 years5.

Tuberculomas constitute 33% of intracranial space-occupying lesions in

patients in developing countries6.

Though CNS tumours constitute only 2 % of all tumours they are very

rapidly progressing lesions causing challenge in accurate diagnosis. The time

of onset to diagnosis plays very crucial role in treating these patients. The

stereotactic biopsies permit accurate sampling of multiple areas and made it

possible to approach hitherto difficult locations. It has increased the

responsibility of pathologist who has to play an important role in the

evaluation, diagnosis and management of these lesions.7

Also non- neoplastic lesions like Tuberculomas, abscesses, fungal

granulomas, and cysts are potentially curable when diagnosed early and

accurately.

NEED FOR THE STUDY

3

NEED FOR THE STUDY

Frozen Section studies are in use since 100 years. Intraoperative cytologic

techniques were introduced in the 1920s. Very few centres continued the use of

smears primarily in the rapid intraoperative diagnosis while other pathologists

preferred frozen sections.

The inherent soft nature and high water content of the nervous tissue

often renders poor quality frozen sections. The advent of stereotactic

procedures has resulted in either supplementing or replacing the use of frozen

section study by the cytology technique in intraoperative consultations1.

Stereotactic biopsies yield tiny tissues and pathologists need a technique

which gives accurate results regarding cellular morphology and at the same

time quick and simple to do8. Hence it is needed to compare the efficacy of

these techniques viz. squash smears and touch imprints with the good old

frozen section study.

AIM AND OBJECTIVES

4

AIM OF THE STUDY

The aim of the study is to assess and compare the utility of squash

smears and frozen sections in intraoperative diagnosis of CNS lesions by

correlating with histopathological examination.

OBJECTIVES OF THE STUDY

To assess the diagnostic utility of squash smears and frozen sections

intraoperatively in CNS lesions

To assess the intraoperative diagnostic accuracy of squash smears and

frozen sections by correlating with histopathology

To compare the results of frozen section and squash smear obtained

intraoperatively with histopathological examination.

To do immunohistochemistry in diagnostically challenging cases.

To determine the limitations of squash smears and frozen sections.

5

REVIEW OF LITERATURE

Space occupying lesions of brain are caused due to tumours, abscesses,

hematoma, cysts (like arachnoid cysts, colloid cysts, dermoid cysts and

epidermoid cysts), tuberculoma, fungal granuloma and rarely cerebral

amoebiasis and cysticercosis. Tumours, primary as well as metastatic, are the

most common etiology of space occupying lesions of brain9. In India, pyogenic

brain abscesses account for approximately 8 to 18 percent of all intracranial

space-occupying masses10.

With the advances in neurosurgical techniques more number of patients

is being operated and it is essential to identify the nature of the lesion

intraoperatively.

It is of paramount importance for the pathologist to know the age, sex,

location of the lesion, whether the lesion is intra-axial or extra-axial and

radiological finding. Other useful information includes the type and duration of

clinical symptoms. For example a history of seizures is suggestive of slow

growing lesions such as low grade tumours whereas signs of elevated

intracranial pressure are associated with high grade tumour. History of previous

surgery, chemotherapy and radiotherapy are also important.

Imaging techniques are the first line investigation in any patient with

CNS symptoms. They include CT, MRI and PET scan (Positron Emission

Tomography). MRI is more sensitive than CT in evaluating posterior fossa

lesions and in detecting infiltration of the cerebral parenchyma. However CT is

superior to MRI in detecting calcified lesions. Cerebral and spinal angiography

are used for vascular lesions.

REVIEW OF LITERATURE

6

Intra-operative diagnosis of brain lesions are needed for the following

reasons4.

1. To confirm that a lesion has been identified;

2. To differentiate neoplastic from reactive lesions;

3. To differentiate metastatic from primary neoplasms;

4. To estimate the degree of malignancy;

5. To determine tumour margins (e.g., Dural margin in meningioma)

6. To obtain tissue for culture or other special procedures; and

7. To ensure safe handling and processing of tissue (e.g., Creutzfeldt-Jakob

disease).

There are basically three techniques to get intraoperative diagnosis of

CNS lesions. They are squash smears, touch imprint and frozen section studies.

Permanent paraffin sections are gold standard.

The proponents of frozen section argue that diagnostic accuracy is

increased due to preserved tissue architecture and similarity to permanent

sections 11, 12, 13. The brain is inherently soft and hence getting a frozen section

is difficult. And it is generally considered that frozen section is good in firm

tumour tissues like meningioma and schwannoma.

With the advent of stereotactic techniques in brain surgery tissues

obtained are very tiny and the pathologist may have to depend entirely upon

cytological features for a definitive diagnosis14. The generally soft consistency

of most primary CNS neoplasms facilitates the preparation of smears, and

smear cytology has been used with great success for the intraoperative

diagnosis of CNS neoplasms 15,16,17,18 .

7

Squash smears and touch imprints are easy to prepare and give a rapid

intraoperative diagnosis with inability to control thickness, crushing artefacts

and difficult smearing are some of the disadvantages of squash smears.

Folkerth 19 detailed the advantages and disadvantages of these

techniques. Touch imprints exhibit better morphological details without any

smearing artefact as compared to squash smears but factors such as poor

cellularity affect the diagnostic accuracy of touch preparation20.

8

SQUASH SMEAR CYTOLOGY

Historical Background

Professor Leonard S. Dudgeon, who was Dean of St. Thomas’s Hospital

Medical School of London, published, together with C.V. Patrick in 1927 21, an

initial study of 200 cases which included some examples of brain tumours. He

popularised the use of so called ‘Wet Film’.

Louise Eisenhardt and Harvey Cushing 22 influenced by him published

an article in 1930 demonstrating the usefulness of the cytologic method for the

rapid diagnosis of brain tumours. The procedure used by these authors was the

performance of squash preparations, stained supravitally with neutral red.

A few years later, supported by this paper, the technique was used

successfully by Russell, Krayenbul, and Cairns in the United Kingdom who

reported their favourable conclusions in 1937.

In 1947, Morris developed a procedure that replaced supra vital stains

with air-dried slides, staining them with eosin and methylene blue dyes.

In 1960, wet fixation in 95 % alcohol was introduced by McMenemey;

this makes possible the use of the more common cytologic stains such as

Haematoxylin-Eosin and Papanicolaou.

The consolidation phase took place during the 1960s and early 1970s.

Marshall and colleagues23 in 1973 first published the accuracy of

diagnosis by cytological techniques. They found that 93.6 % of 184 specimens

were diagnosed correctly by intraoperative cytology.

9

ADVANTAGES and DISADVANTAGES 4,18,24,25

Advantages of the Smear Technique

More rapid and technically simpler

Only a small amount of tissue needed

Far better preservation of cellular detail

No freezing artefact

It allows evaluation of the background

It allows a more thorough sampling (multiple biopsies from a large

lesion can easily be examined)

It allows the study of tissues that are difficult to section, particularly

those that are necrotic & calcified. If the case turns out to be infectious,

contamination of the cryostat and subsequent defrosting and sterilization

can be avoided

Disadvantages of the Smear Technique

Some architectural details are lost

Some lesions do not smear well- they are not very useful in cohesive

lesions like schwannomas, meningioma 26 and metastatic lesions

Not useful for evaluating the margin status 27,28,29

10

FROZEN SECTION STUDY

Historical Background:

The history of the frozen section as intraoperative consultation is

intertwined with the development of pathology as a clinical specialty.

William Halsted, Chief of Surgery of the new Johns Hopkins hospital,

requested the first intraoperative frozen section be done by the pathologist

William Welch in 1891. But before Welch completed the study Halsted

finished the operation.

Thomas Cullen published a frozen section method in the Johns Hopkins’

bulletin in 1895. The procedure still took nearly an hour to complete.

It is generally accepted that the standard cryostat method used today was

first published in JAMA in 1905 by Louis Wilson 30 of the Mayo Clinic. Wilson

used a dextrin solution to embed the tissue and a carbon dioxide freezing

microtome. By using methylene blue and reading the slides without permanent

mounting the technique could be performed in a few minutes as opposed to an

hour for Cullen’s method. This method rapidly became a routine at the Mayo

Clinic.

A strong proponent of the frozen section was Dr Joseph Bloodgood who

began a campaign to promote the frozen section as a medical standard. He

submitted an editorial in JAMA in 1927 31, extended the invitation to surgeons

and pathologists to visit the laboratory at Johns Hopkins.

11

The acceptance of the frozen section as a diagnostic tool in the setting of

a one-step surgical procedure eventually led to the acceptance of biopsy

techniques to allow a pre-surgical diagnosis.

The frozen section played a pivotal role in the acceptance of microscopy

in the clinical management of patients and the development of modern surgical

pathology.

12

ADVANTAGES and DISADVANTAGES1

Advantages of Frozen Section Study

Provides better architectural details 32,33

Better in diagnosing in firmer lesions like meningioma, schwannoma

Delineating margin status

Good old technique

Disadvantages of Frozen Section Study

Freezing artefact – nervous tissues as they are soft and has high water

content reduces the quality frozen section and ice crystal artefact occur

due to freezing

Need of expensive equipment34,35

Technical expertise needed

Requires electricity

13

INTERPRETATION OF SMEAR PREPARATION

Before attempting to interpret the smear it is essential to know the normal

cytology36 of CNS in smear preparation.

White Matter Pattern

Normal white matter produces evenly distributed smears.

The background is eosinophilic and finely granular.

There is a low cellularity consisting basically of oligodendrocytes and to a

lesser extent of astrocytes.

The cytological discrimination between these two normal glial cells is difficult,

but not necessary.

Vascularization consists of fine capillaries with elongated endothelial cells

arranged parallel to the capillary wall.

Grey Matter Pattern

Tissue tends to smear less evenly than tissue from white matter,

accumulating elements of larger size, such as blood vessels and neurons, at the

edge of the smear.

The background is also eosinophilic and granular, but cellularity is always

higher than that in white matter.

Together with the glial cells numerous neurons appear as large cells with

abundant cytoplasm and distinct cell borders and round, prominent nucleolated

nuclei.

Capillaries are particularly distinctive, but the smears will also contain

arterioles and small arteries.

14

Because of higher cellularity, low-grade gliomas and glio-neural

tumours are to be included in the differential diagnosis of this pattern.

Cerebellar Cortex Pattern

The predominant cells are neurons from the internal granular layer,

which look like small, round, hyperchromatic cells without visible cytoplasm.

In this ocean of small cells, there are islands of large Purkinje neurons.

.

Due to the predominance of neurons from the internal granular layer, this

type of smear must be differentiated from smears originating from embryonal

tumours, small cell carcinomas, and lymphomas.

Choroid Plexus Pattern

The choroid plexus tends to smear poorly, remaining as a cohesive crush

preparation. Its vessels show a complex arched papillary appearance. The cells

appear isolated and show a wide polygonal cytoplasm and a small round central

nucleus.

This differentiates them from the smears of choroid plexus papilloma

which are highly cellular and show papillae, monolayers, tridimensional

aggregates, and single cells.

Leptomeningeal Pattern

The cytoplasm is wide and delicate and their nuclei are uniform and

ovoid and with finely granular chromatin. Arachnoidal cells have poorly

defined boundaries, giving rise to sheets with a syncytial appearance. There is a

tendency to have a whorl-like cellular arrangement, which is why they may be

confused with meningioma.

15

Reactive Changes In Smear Preparations

Reactive Gliosis

Gliosis usually smears very poorly, whereas most astrocytoma smears

much more easily. The background is clearly fibrillary in the case of a tumour.

In gliosis it is the finely granular and eosinophilic background of the normal

brain.

Against this background, reactive astrocytes tend to remain in small

groups, showing scant-to abundant eosinophilic cytoplasm, with coarse

cytoplasmic processes and minimal, if any, nuclear atypia. Correlation with

clinical-radiologic aspects is essential.

Comparison Between Gliosis and Astrocytoma

16

Reactive Microglia

They are seen as rod cells in smears with elongated nuclei and scanty

cytoplasm

Acute Inflammatory Conditions

Acute inflammatory cells mainly polymorphs are seen in abscesses, early

stages of infarction and tumour necrosis

Chronic inflammatory conditions

They are characterised by perivascular lymphocytic cuffing which are

very well demonstrated in squash smears. Multi nucleated giant cells and

epithelioid cells are seen in tuberculous infection. In cryptococcal infection

there are numerous macrophages which can occasionally contain the organism.

ARTEFACTS

They can arise at several stages in preparation and staining of smears.

They can hinder the diagnosis by distorting the cytological features.

Artefact Mistaken Interpretation

Crush artefact Fibrillary astrocytoma

Thick smear Malignant glioma

Bone dust Calcospherites

Starch powder Psammoma bodies

Haemostatic sponge Calcification

Drying artefact Necrosis/Mitosis /Apoptosis

17

INTERPRETATION OF FROZEN SECTION 3

White matter:

White matter is characterized by cells with rounded nuclei and scant

cytoplasm (oligodendrocytes). A smaller population of cells with larger nuclei

(astrocytes) are also seen. Neurons are generally not observable in the white

matter.

Grey Matter

Grey matter tissue is characterized by the presence of neurons. Many of

the larger size neurons have prominent nucleoli in their round to oval nuclei.

Nissl substance is seen in their cytoplasm.

Cerebellum

Cerebellum shows the three layers of cortex, including the paucicellular

molecular layer, Purkinje cell layer, and granular cell layer. A biopsy taken

from the granular cell layer only may be misinterpreted as representing chronic

inflammation

Infant brains may show an additional granular cell layer at the surface.

Meninges

The meninges comprise loose connective tissue with blood vessels, nests

of meningothelial or arachnoidal cap cells and occasional nevus cells, which

are spindled with melanin pigment.

Pineal Gland

The pineal gland is marked by rounded or slightly elongated cells

arranged in lobules, separated by fibrovascular tissue. The background of the

18

gland has a fibrillary appearance. Micro calcifications are also a frequent

finding in the pineal gland in adults.

Pituitary Neurohypophysis

The pituitary neurohypophysis is marked by a loose arrangement of

spindled cells, resembling a spindled glioma.

Reactive Changes

Reactive astrocytes in gliosis are marked by increased eosinophilic

cytoplasm and mild nuclear enlargement with displacement of the nucleus to

the periphery of the cell. The nuclei may show a round to oval or kidney bean

shaped contour.

The appearance of reactive astrocytes contrasts with typical malignant

astrocytes which are marked by high nuclear cytoplasmic ratio, angular and

irregular nuclear contours, and nuclear hyperchromasia. Brightly eosinophilic

Rosenthal fibres may be seen in areas of gliosis.

Cytology of Certain CNS Lesions

Fibrillary astrocytoma

It is characterised by dense fibrillary background with increased

cellularity and mild nuclear atypia. The cytoplasm is not discernible.

Capillaries are the characteristic vessels of low-grade astrocytoma.

Gemistocytic astrocytoma

It is characterised by large cells with eccentric nuclei and plump, glassy

eosinophilic cytoplasm. There are numerous and coarse cytoplasmic processes.

19

Protoplasmic astrocytoma

It is characterised by an evenly distributed smear with small cells

showing round-to-oval nuclei and sparse cytoplasmic processes.

Anaplastic Astrocytoma

It is characterised by a dense fibrillary background with increased

cellularity and nuclear atypia. Gemistocytic cellular component is a common

feature of anaplastic astrocytoma.

Glioblastoma

The background may be fibrillary, necrotic or necrotic-fibrillary.

Presence of necrosis is of great value. Cells may be atypical astrocytic cells,

sometimes giant multinucleated cells.

The chromatin is coarse and irregular nuclear membrane frequently

shows numerous folds and deep indentations that give the nucleus a multi lobed

appearance. Intercellular cohesiveness is very low, and, the hence tumour cells

appear as isolated elements or form, at most, small loose groups.

Microvascular proliferation (“glomeruloid tufts”), medium- and

small-size vessels with intraluminal endothelial proliferation, and vascular

thrombosis may be seen.

20

Pilocytic Astrocytoma

Bipolar cells with bland nuclei and Rosenthal fibres are seen in a

fibrillary background. Characteristic piloid cells with very long “hair-like”

processes admixed with protoplasmic-like cells are seen.

Pilomyxoid Astrocytoma

The characteristic findings include monomorphous piloid cells and mixed

fibrillary-myxoid background.

Oligodendroglioma

It is characterised by uniform population of isolated small round cells

without cytoplasmic processes. The background is granular-vacuolated;

cytoplasm is scant and ill-defined with no peri nuclear halos. This artefact is

caused by delayed fixation and is not seen in smears.

“chicken-wire” capillary network is characteristic. Micro calcification may

be seen.

Ependymoma

It is characterised by fibrillary background with clumps of small cells

with distinct dual, glial-epithelial features seen with round-to-oval dark nuclei

with evenly distributed granular chromatin.

21

Central neurocytoma

It is characterised by a highly cellular smear with a diffuse pattern of

round, small isomorphic cells with neurocytic rosettes.

Medulloblastoma

Diffuse pattern of non-cohesive dark blue cells seen. The most defining

cytologic features are marked hyperchromasia, high nucleus/cytoplasm ratios,

and an undifferentiated aspect. Nuclei are round-to-oval with coarse chromatin.

Meningioma

Discohesive clumps of uniform cells exhibiting a copious, wispy, and

folded cytoplasm are characteristic. Cell groups have syncytial appearance with

ill-de fined cellular boundaries. Nuclei are slightly oval and some have well-de

fined cytoplasmic invaginations or pseudo inclusions. The presence of cell

whorls is a key finding.

Psammomatous meningioma

Cellular smear showing many calcified Psammoma bodies.

Fibrous meningioma

It is characterised by smear showing fusiform cells with tapering

cytoplasmic processes. Nuclei show the characteristic meningothelial features,

including nuclear cytoplasmic pseudo inclusions.

22

Atypical meningioma

Crowded sheets of meningothelial atypical cells with enlarged and

hyperchromatic nuclei.

Hemangioblastoma

Vascular core tissue fragment with associated coarse stromal cells having

the characteristics intracytoplasmic lipidic vacuoles.

Schwannomas

All nerve sheath tumours tend to be very resistant to disaggregation and

difficult to crush.

Antoni A areas show cluster composed of tightly packed spindle cells

without visible cytoplasmic borders long, club-shaped nuclei with tapered end.

Antoni B areas show loose cluster composed by less regular cells

exhibiting round nuclei.

Pineocytoma

Discohesive pattern of small and remarkable uniform, round cells with

irregular and confluent pineocytomatous rosettes are seen.

Pituitary adenoma

Discohesive pattern of abundant small cells with numerous bare nuclei,

speckled chromatin and small well-defined nucleoli are seen. Vascular-core

papillary structures with tumour cell aggregations are seen. Many cells exhibit

a plasmocytoid feature with eccentric nuclei.

23

Cytology of Metastatic Carcinomas:

Cellular smears

Cohesive clumps of cells

High nuclear/cytoplasmic ratio

Distinct cell borders

No cytoplasmic processes

No fibrillary background

Adenocarcinoma may be recognized by the presence of papillary

structures, 3-dimensional groups, and intra- or extracellular mucin.

Squamous cell carcinoma may show whorls, individual keratinization, and

dense orangeophilic cytoplasm

Small cell carcinoma shows small size, nuclear moulding, and crush

artefact.

Renal cell carcinomas often display sheets of large cells with clear

multivacuolated cytoplasm and macro nucleoli.

Breast carcinoma is characterized by of intracellular lumina containing

mucin secretion.

Colonic carcinoma usually has columnar cells with elongated and stratified

nuclei.

24

Urothelial cell carcinoma often displays flattened sheets of cells with

homogeneous cytoplasm and isolated cells with elongated unipolar cytoplasmic

processes and a blunt pole that contains the nucleus (cercariform cells).

Melanoma typically smears out as individual cells with epithelioid or

fusiform features. Dark-brown granular cytoplasmic pigment is extremely

helpful.

Small “ blue ” cell sarcomas of childhood (embryonal

rhabdomyosarcoma and Ewing sarcoma) display a single cell pattern of small,

hyperchromatic round cells in a striped background.

Epidermoid Cysts

Smears from the cyst contents show clusters of anucleate squames,

occasionally with cytoplasmic keratohyaline granules.

Tuberculosis

Multinucleated giant cell, histiocytes, and inflammatory cells in a

necrotic background are seen.

25

Frozen Section Study Of Certain CNS Lesions

PRIMARY TUMOURS:

Tumour grade should be made at the time of frozen section consultation

that is preliminary but final grading is based on the evaluation of the permanent

sections.

Pilocytic Astrocytoma (Grade I) is characterized by typical biphasic

appearance marked by a more densely fibrillary zone alternating with an area

that has a looser, microcystic appearance. Rosenthal fibres, eosinophilic

granular bodies, and occasional pleomorphic nuclei are also seen.

In paraffin section a similar bipolar fibrillary cells admixed with areas of

stellate cells with short processes are seen.

Grade II astrocytoma is marked by mild hyper cellularity and an uneven

distribution of atypical cells.

Grade III astrocytoma is usually marked by increased cellularity and nuclear

atypia, and mitosis. In paraffin section similar features seen.

A glioblastoma is characterized by the presence of either vascular proliferation

and/or necrosis, which may or may not be rimmed by a pseudo palisade of

tumour cells.

In paraffin section similar features seen.

26

Oligodendroglioma

Distinction of Oligodendroglioma from astrocytoma at frozen section

may be nearly impossible. It is not essential to distinguish between an

Oligodendroglioma and an astrocytoma at the time of frozen section

consultation. A diagnosis of a low-grade or a high-grade glioma is adequate.

Oligodendroglioma tend to have a more monomorphic appearing

population of cells with generally round nuclei. The classic pericellular

clearing, represents an artefact of delayed formalin fixation and is not evident

at frozen section.

Micro calcifications are a common feature of many oligodendrogliomas

(up to 80% of tutors) but are not diagnostic of oligodendrogliomas. They have

a prominent capillary vascular pattern due to the paucity of cellular processes

in the tumour cells.

Paraffin section shows artefactual perinuclear cytoplasmic clearing

“Fried Egg appearance”. Abundant vascular stroma forming network of thin

walled blood vessels mimicking chicken wire pattern is seen.

Meningioma

Meningothelial meningioma (WHO Grade I) is marked by a whorling

pattern and characteristic intranuclear pseudo inclusions.

Fibrous meningioma (WHO Grade I ) is marked by a proliferation of

bland appearing spindled cells.

Prominent numbers of Psammoma body calcifications mark the

psammomatous

variant of meningioma (WHO Grade I)

27

Atypical meningioma(WHO Grade II) is characterized by the presence

of 4 or more mitotic figures per 10 high power fields; brain invasion; or a

constellation of three or more of the following features, including increased

cellularity, small cell change, prominent nucleolation, loss of lobular

architecture, and necrosis.

Rare anaplastic meningioma (WHO Grade III) demonstrates evidence of

20 or more mitoses per 10 high power fields or overt anaplasia.

Differential diagnosis of cerebellopontine angle lesions

Schwannoma

Meningioma

Epidermoid cyst

Ependymoma

Choroid plexus tumour

Schwannoma (WHO Grade I)

A schwannoma is characterised by sclerotic blood vessels, Antoni A and

Antoni B patterns and verocay bodies.

The Antoni A pattern may be confused with a fibrous meningioma.

The Antoni B pattern may mimic a low-grade glioma.

28

Ependymoma (WHO Grade II)

A low-grade ependymoma marked by the presence of perivascular pseudo

rosettes. There must be either pseudo rosettes or true ependymal rosettes to

make a definitive diagnosis of ependymoma at the time of frozen section.

If there are no discernible rosettes a differential diagnosis of infiltrating

astrocytoma may be confused with and in such cases, a frozen section

diagnosis of a low-grade glioma is adequate.

Myxopapillary ependymomas (WHO Grade I) are marked by blood

vessels surrounded by a mucinous stroma, which is in turn surrounded by

tumour cells. Rosettes and pseudo rosettes are generally lacking. Similar

features are seen in paraffin section.

Central Neurocytoma (WHO Grade II)

Central neurocytoma is marked by a sheet of monomorphic appearing

cells arranged against fibrillary background.

Medulloblastoma (WHO Grade IV)

Medulloblastoma is characterized by a dense population of cells with

generally high nuclear to cytoplasmic ratios. Apoptosis, geographic necrosis,

and increased mitotic activity are common. In a minority of cases, Homer

Wright pseudo rosettes may be seen.

Hemangioblastoma (WHO Grade I)

The hemangioblastoma is marked by an increased number of blood

vessels with admixed stromal cells.

29

Pituitary Adenoma

It is marked by a sheet-like proliferation of monomorphic appearing cells

with discohesion.

Pineocytoma

It is marked by the presence of pineocytomatous pseudo rosettes in

which tumour cells are arranged around a fibrillary core. In their absence it is

difficult to differentiate from normal pineal gland tissue.

Metastatic Tumours

The most common sites of origin include lung, breast, kidney, skin

(melanoma), and gastrointestinal tract. In many cases, the tumour is so poorly

differentiated that discerning the specific tumour type is not always possible,

particularly in the frozen section venue.

Granulomatous inflammation

The presence of granulomatous inflammation associated with necrosis

should raise the suspicion of a possible infections such as tuberculosis and

fungal infections.

Epidermoid cyst

It is lined by stratified squamous epithelium with keratohyaline granules.

30

WHO CLASSIFICATION AND HISTOLOGICAL GRADING OF CNS

TUMOURS 37(ANNEXURE)

The 2000 and 2007 WHO classifications considered histological features

along with the rapidly increasing knowledge of the genetic changes that

underlie the tumorigenesis of CNS tumours.

The present update (the 2016 classification) breaks with this nearly

century-old tradition and incorporates well-established molecular parameters

into the classification of diffuse gliomas. An important consideration for the

2016 WHO classification was that the implementation of combined

phenotypic- genotypic diagnostics in some large centres with the increasing

availability of immuno histochemical surrogates.

Many of the genetic parameters included in the 2016 WHO classification

can be assessed using immunohistochemistry or FISH.

Grade I lesions are generally tumours with low proliferative potential and the

possibility of cure after surgical resection alone.

Grade II lesions are usually infiltrative in nature and often recur, despite having

low levels of proliferative activity.

The Grade III designation is applied to lesions with clear histological evidence

of malignancy, including nuclear atypia and sometimes brisk mitotic activity.

In most settings, patients with grade III tumours receive radiation and/or

chemotherapy.

The Grade IV (Glioblastoma) designation is applied to cytologically malignant,

mitotically active, necrosis-prone neoplasms that are often associated with

rapid pre- and post -operative disease evolution and fatal outcome.

31

MATERIALS AND METHODS

This is a prospective study conducted at institute of pathology, Madras

Medical College& Rajiv Gandhi Government General Hospital, Chennai in

coordination with Department of Neuro surgery after getting institutional

ethical committee clearance.

This study compares the diagnostic accuracy and limitations of Squash

Smear Examination and Frozen Section Study in the intraoperative diagnosis of

space occupying lesions of central nervous system in relevance with

histopathological examination.

The duration of study was one year from 1st August 2016 to 31st August

2017.

The total number of patients included in the study was 50. They were

patients presenting to Department Of Neurosurgery, Madras Medical College

& Rajiv Gandhi Government General Hospital, Chennai and had been

diagnosed with space occupying lesions in the central nervous system by

clinical and radiological examination and were posted for surgery and where a

request was made by the operating neurosurgeon regarding intraoperative

diagnosis.

Inclusion Criteria:

Those patients posted for surgery for a space occupying lesion in the

central nervous system and where a request had been made by the operating

neurosurgeon for an intraoperative diagnosis.

MATERIALS AND

METHODS

32

METHODS

The tissue obtained from the neurosurgeon intraoperatively was subjected

to squash smear examination and frozen section examination.

An intraoperative diagnosis was made and surgery proceeded

accordingly. The tissue obtained at the end of the procedure was subjected to

histopathological examination and the results of squash smear, frozen section

and histopathological examination were compared and analysed. The method

of squash smear preparation and examination and frozen section examination

are as detailed below.

Method of Squash Smear Preparation and Examination:

The tissue obtained was carried from the operating room to

neuropathology laboratory in a sterile container on saline moistened gauze to

prevent drying accompanied by request form which summarized relevant

clinical and radiological data including the precise site of biopsy and reason for

which intraoperative diagnosis was sought.

After receiving the specimen a gross inspection of the same was made.

For squash smear preparation a tiny bit of tissue measuring 0.5 to 1 sq. mm

without necrotic and haemorrhagic areas was taken and placed at one end of a

labelled clean glass slide and smear was made by compressing the tissue with a

second slide and drawing the second slide quickly across the first slide and a

relatively uniform tissue layer was produced. Care was taken to exert a right

amount of pressure on to the slide.

The smear preparation was fixed immediately while still moist in isopropyl

alcohol for five minutes and then it was stained with rapid haematoxylin and

eosin stain.

33

Procedure of rapid staining:

The smear was stained in haematoxylin for five minutes and washed in running

tap water till sections turned blue followed by dipping in 1% acid alcohol for

differentiation for five seconds and then washed in water. It was later stained in

eosin for a minute and washed in water followed by absolute alcohol and

cleared in Xylol. It was mounted with DPX. The time taken to complete the

staining procedure was approximately 8 to 10 minutes.4, 35,38,39,40

Method of Frozen Section Study:

The tissue was processed in cryostat at −10 degree C to −15 degree C.

Thin sections of 3-5 μm thickness were cut and fixed immediately in isopropyl

alcohol and rapid H & E staining done.

The section was stained in haematoxylin for 2 minutes and then washed

in running tap water, dipped in 1% acid alcohol for differentiation and washed

under running tap water. Then the slide was dipped in aqueous ammonia

solution for bluing for few seconds followed by staining in eosin for 30

seconds.

Then the section was dehydrated with absolute alcohol by giving 5 dip

each in two jars of alcohol followed by pressing the slide between filter paper

and clearing done with xylene by giving 5 dip each in two jars of xylene

followed by pressing the slide between filter paper and finally mounted in

DPX.

34

The results of squash smear study and frozen section studies were

immediately intimated to the operating neurosurgeon and the operative

procedure was completed accordingly. The postoperative specimen was then

sent in 10% neutral buffered formalin for histopathological examination. The

results of squash smear, frozen section and HPE were then compared and

analysed. In case of tumours, grading was done as per WHO and correlated

with HPE grading.

In difficult cases where conclusion could not be made even with

histopathology special stains and immunohistochemistry were used as

adjuvants to histopathology. Whenever there was difficulty in grading

astrocytoma, tumour grading was done with Ki67/MIB-1 labelling index using

immunohistochemistry.

The results of squash smear cytology (A), frozen section study (B) were

correlated with histopathological examination (C).

RESULTS

35

RESULTS

The results derived from the study are as follows

Table 1 Age Distribution

Frequency Percent Valid Percent

Cumulative

Percent

Valid

Less than 1 Year 1 2.0 2.0 2.0

1 -5 Years 1 2.0 2.0 4.0

6 - 10 Years 4 8.0 8.0 12.0

11 - 20 Years 4 8.0 8.0 20.0

21 - 30 Years 6 12.0 12.0 32.0

31 - 40 Years 7 14.0 14.0 46.0

41 - 50 Years 15 30.0 30.0 76.0

51 - 60 Years 8 16.0 16.0 92.0

More than 60 Years 4 8.0 8.0 100.0

Total 50 100.0 100.0

36

The youngest patient in our study was aged 6 months and the eldest one

was 70 years of age. Majority of the patients were in their fourth decade. The

median age was 42 years.

2% 2%

8%

8%

12%

14%30%

16%

8%

Age Group

Less than 1 Year 1 -5 Years

6 - 10 Years 11 - 20 Years

21 - 30 Years 31 - 40 Years

41 - 50 Years 51 - 60 Years

More than 60 Years

37

Table 2-Sex Distribution

Frequency Percent Valid Percent

Cumulative

Percent

Valid Male 26 52.0 52.0 52.0

Female 24 48.0 48.0 100.0

Total 50 100.0 100.0

In our study out of 50 patients 26(52%) were males and 24(48%) were females.

0

10

20

30

40

50

60

Male Female

5248

Sex

38

Table 3-Provisional Diagnosis

Frequency Percent Valid Percent

Cumulative

Percent

Valid Low grade glioma 8 16.0 16.0 16.0

High grade glioma 11 22.0 22.0 38.0

Small round cell tumour 5 10.0 10.0 48.0

Meningioma 7 14.0 14.0 62.0

Schwannoma 4 8.0 8.0 70.0

Pituitary Adenoma 1 2.0 2.0 72.0

Metastasis 3 6.0 6.0 78.0

Hemangioblastoma 1 2.0 2.0 80.0

Epidermoid Cyst 1 2.0 2.0 82.0

Granulomatous Lesion 3 6.0 6.0 88.0

Ependymoma 3 6.0 6.0 94.0

Others 3 6.0 6.0 100.0

Total 50 100.0 100.0

39

In our study of the 50 patients most common clinico radiological was

glioma (19 patients) and the next common diagnosis was meningioma (7

patients). Only one patient was diagnosed with pituitary adenoma.

16%

22%

10%14%

8%

2%

6%

2%2%

6%

6%6%

Provisional Diagnosis

Low grade glioma High grade glioma Small round cell tumour

Meningioma Schwannoma Pituitary Adenoma

Metastasis Hemangioblastoma Epidermoid Cyst

Granulomatous Lesion Ependymoma Others

40

Table 4-Site of Lesion

Frequency Percent Valid Percent

Cumulative

Percent

Valid Frontal 20 40.0 40.0 40.0

Temporal 4 8.0 8.0 48.0

Parietal 3 6.0 6.0 54.0

Cerebello Pontine Angle 3 6.0 6.0 60.0

Sellar/Suprasellar 1 2.0 2.0 62.0

Thalamus 1 2.0 2.0 64.0

Corpus Callosum 3 6.0 6.0 70.0

Brain Stem 3 6.0 6.0 76.0

Cerebellum 6 12.0 12.0 88.0

Ventricle 4 8.0 8.0 96.0

Base Of Skull 1 2.0 2.0 98.0

Spine 1 2.0 2.0 100.0

Total 50 100.0 100.0

41

Of the total 50 patients studied, in 20 patients (40%) frontal lobe was

involved and cerebellum was the second common site which was involved in 6

patients (12%). Cerebellopontine angle lesions comprised 6% of cases. We also

had one patient (2%) with spinal lesion.

40%

8%6%

6%

2%2%

6%

6%

12%

8%2% 2%

Site Of Lesion

Frontal Temporal Parietal

Cerebello Pontine Angle Sellar/Suprasellar Thalamus

Corpus Callosum Brain Stem Cerebellum

Ventricle Base Of Skull Spine

42

Table 5-Neoplastic Vs Non Neoplastic

Frequency Percent Valid Percent Cumulative

Percent

Valid

Yes 45 90.0 90.0 90.0

No 5 10.0 10.0 100.0

Total 50 100.0 100.0

43

As many as 45 patients (90%) were diagnosed with neoplastic

lesions in our study.

0

20

40

60

80

100

Yes No

90

10

Neoplastic

44

Table 6-Results of Squash Smear Cytology

Frequency Percent Valid Percent

Cumulative

Percent

Valid Low grade glioma 12 24.0 24.0 24.0

High grade glioma 7 14.0 14.0 38.0

Small round cell tumour 5 10.0 10.0 48.0

Meningioma 6 12.0 12.0 60.0

Schwannoma 3 6.0 6.0 66.0

Pituitary Adenoma 1 2.0 2.0 68.0

Metastatic Carcinomatous

Deposits

3 6.0 6.0 74.0

Hemangioblastoma 1 2.0 2.0 76.0

Epidermoid Cyst 2 4.0 4.0 80.0

Granulomatous Lesion 3 6.0 6.0 86.0

Malignant Melanoma 1 2.0 2.0 88.0

Organised Hematoma 1 2.0 2.0 90.0

Ependymoma 5 10.0 10.0 100.0

Total 50 100.0 100.0

45

After running the squash smear cytology test we found out that majority

of the lesions turned out to be gliomas of which low grade gliomas were

12(24%) and high grade gliomas were 7(14%). There were 6 (12%) cases of

meningioma and 5 (10%) cases each of ependymoma and small round cell

tumour. Metastasis contributed to 6%(3 cases) of the CNS lesions in our study

and the same number of patients was diagnosed with granulomatous lesions.

24%

14%

10%12%

6%

2%

6%2%

4%

6%2%2%

10%

Squash Smear Cytology

Low grade glioma High grade glioma

Small round cell tumour Meningioma

Schwannoma Pituitary Adenoma

Metastatic Carcinomatous Deposits Hemangioblastoma

Epidermoid Cyst Granulomatous Lesion

Malignant Melanoma Organised Hematoma

Ependymoma

46

Table 7- Results of frozen section study

Frequency Percent Valid Percent Cumulative Percent

Valid Low grade glioma 12 24.0 24.0 24.0

High grade glioma 9 18.0 18.0 42.0

Small round cell tumour 4 8.0 8.0 50.0

Meningioma 6 12.0 12.0 62.0

Schwannoma 3 6.0 6.0 68.0

Pituitary Adenoma 1 2.0 2.0 70.0

Metastatic Carcinomatous

Deposits

3 6.0 6.0 76.0

Hemangioblastoma 1 2.0 2.0 78.0

Epidermoid Cyst 2 4.0 4.0 82.0

Granulomatous Lesion 3 6.0 6.0 88.0

Malignant Melanoma 1 2.0 2.0 90.0

Ependymoma 5 10.0 10.0 100.0

Total 50 100.0 100.0

47

By frozen section study 12 patients (24%) were diagnosed with low grade

glioma and 9 patients (18%) were having high grade glioma. Out of 50 patients

3 patients (6%) were diagnosed to have metastatic carcinomatous deposits and

3 patients (6%) with granulomatous lesions. Twelve percent of the lesions

turned out to be meningioma.

22%

16%

7%11%

6%2%

5%2%4%

5%2%

9%

9%Frozen Section Study

Low grade glioma High grade glioma

Small round cell tumour Meningioma

Schwannoma Pituitary Adenoma

Metastatic Carcinomatous Deposits Hemangioblastoma

Epidermoid Cyst Granulomatous Lesion

Malignant Melanoma Ependymoma

Ependymoma

48

Table 8-Resuls of Histopathology

Frequency Percent Valid Percent Cumulative Percent

Valid Low grade glioma 10 20.0 20.0 20.0

High grade glioma 10 20.0 20.0 40.0

Small round cell tumour 5 10.0 10.0 50.0

Meningioma 6 12.0 12.0 62.0

Schwannoma 3 6.0 6.0 68.0

Pituitary Adenoma 1 2.0 2.0 70.0

Metastatic Carcinomatous

Deposits

3 6.0 6.0 76.0

Hemangioblastoma 1 2.0 2.0 78.0

Epidermoid Cyst 2 4.0 4.0 82.0

Granulomatous Lesion 3 6.0 6.0 88.0

Malignant Melanoma 1 2.0 2.0 90.0

Ependymoma 5 10.0 10.0 100.0

Total 50 100.0 100.0

49

We had done histopathology examination in all the 50 patients. Among

the 50 patients 20 patients (40%) were diagnosed to have gliomas. Among the

gliomas 10 (20%) were low grade gliomas that included Pilocytic astrocytoma,

diffuse astrocytoma, mixed oligoastrocytoma and Oligodendroglioma. The

remaining ten gliomas were high grade gliomas (20%) that included

glioblastoma multiforme, anaplastic astrocytoma and anaplastic

oligodendroglioma.

As many as 6 patients (12%) were having meningioma, 5 patients (10%)

were having ependymoma, and 3 patients (6%) were having metastatic

carcinomatous deposits and 3 patients (6%) with granulomatous lesions.

As many as 5 patients (10%) were having small round cell tumours which

included Medulloblastoma and Esthesioneuroblastoma.

18%

18%

9%11%

6%2%

5%

2%4%

5%2%

9%

9%

Histopathology

Low grade glioma High grade glioma

Small round cell tumour Meningioma

Schwannoma Pituitary Adenoma

Metastatic Carcinomatous Deposits Hemangioblastoma

Epidermoid Cyst Granulomatous Lesion

Malignant Melanoma Ependymoma

Ependymoma

50

Table 9-Comparison between Squash Smear Cytology (A), Frozen Section

Study (B) and Histopathological Examination(C)

Frequency Percent Valid

Percent

Cumulative

Percent

Valid A, B & C reveal same diagnosis 41 82.0 82.0 82.0

A & C reveal same diagnosis but B is

different 1 2.0 2.0 84.0

B & C reveal same diagnosis but A is

different 3 6.0 6.0 90.0

A & B reveal same diagnosis but C is

different 4 8.0 8.0 98.0

A , B & C are all different 1 2.0 2.0 100.0

Total 50 100.0 100.0

51

In as many as 41 patients (82%), squash smear cytology (A), frozen

section study (B) and Histopathological examination (C) revealed the same

diagnosis. In one patient (2%) , squash smear cytology differed from frozen

section study but compared with histopathological examination. In three

patients (6%), squash smear cytology (A) differed from frozen section study

(B) where frozen section study compared with histopathology (C). In four

patients (8%), histopathological examination (C) was different from frozen

section study (B) as well as squash smear cytology (A) when the latter two

showed same diagnosis.

In only one patient, Immunohistochemistry was used to get final

diagnosis where (A), (B) and (C) were all different.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

A,B& C SameDiagnosis

A & C Same BDifferent

B & C Same ADifferent

A & B Same CDifferent

A,B& C Are allDifferent

52

Table -10 Correlation between Squash Smear Cytology (A) &Histopathological Examination (C)

Histopathology (C)

To

tal

Low

gra

de g

liom

a

Hig

h g

rade g

liom

a

Sm

all

round c

ell

tum

our

Me

nin

gio

ma

Schw

annom

a

Pituitary

Adenom

a

Me

tasta

tic C

arc

inom

ato

us

Deposits

Hem

angio

bla

sto

ma

Epid

erm

oid

Cyst

Gra

nulo

ma

tous L

esio

n

Ma

lignant M

ela

nom

a

Ependym

om

a

Squash Smear Cytology(A) Low grade glioma Count 8 3 1 0 0 0 0 0 0 0 0 0 12

% within Squash Smear Cytology(A) 66.7% 25.0% 8.3% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

High grade glioma Count 0 6 1 0 0 0 0 0 0 0 0 0 7

% within Squash Smear Cytology(A) .0% 85.7% 14.3% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Small round cell tumour Count 2 0 3 0 0 0 0 0 0 0 0 0 5

% within Squash Smear Cytology(A) 40.0% .0% 60.0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Meningioma Count 0 0 0 6 0 0 0 0 0 0 0 0 6

% within Squash Smear Cytology(A) .0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Schwannoma Count 0 0 0 0 3 0 0 0 0 0 0 0 3

% within Squash Smear Cytology(A) .0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Pituitary Adenoma Count 0 0 0 0 0 1 0 0 0 0 0 0 1

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% 100.0%

53

Metastatic Carcinomatous Deposits Count 0 0 0 0 0 0 3 0 0 0 0 0 3

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% 100.0%

Hemangioblastoma Count 0 0 0 0 0 0 0 1 0 0 0 0 1

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% 100.0%

Epidermoid Cyst Count 0 0 0 0 0 0 0 0 2 0 0 0 2

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% 100.0%

Granulomatous Lesion Count 0 0 0 0 0 0 0 0 0 3 0 0 3

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% 100.0%

Malignant Melanoma Count 0 0 0 0 0 0 0 0 0 0 1 0 1

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% 100.0%

Organised Hematoma Count 0 1 0 0 0 0 0 0 0 0 0 0 1

% within Squash Smear Cytology(A) .0% 100.0% .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Ependymoma Count 0 0 0 0 0 0 0 0 0 0 0 5 5

% within Squash Smear Cytology(A) .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% 100.0%

Total Count 10 10 5 6 3 1 3 1 2 3 1 5 50

% within Squash Smear Cytology(A) 20.0% 20.0% 10.0% 12.0% 6.0% 2.0% 6.0% 2.0% 4.0% 6.0% 2.0% 10.0% 100.0%

54

The above table correlates the finding in squash smear cytology with

histopathological examination. There was one hundred percent correlation in the case

of meningioma, schwannoma, hemangioblastoma, ependymoma, pituitary adenoma,

metastatic carcinomatous deposits, epidermoid cyst, malignant melanoma and

granulomatous lesions. In the case of low grade glioma it was 66.7% and in high

grade glioma it was 85.7 %.

66.7

85.7

60

100 100 100 100 100 100 100 100 100 100

0

20

40

60

80

100

120

Correlation between Squash Smear Cytology and Histopathology

55

Chi-Square Tests

Value df Asymp. Sig. (2-

sided)

Pearson Chi-Square 485.393a 132 .000

Likelihood Ratio 190.615 132 .001

Linear-by-Linear Association 42.173 1 .000

N of Valid Cases 50

The p value in this observation was .000 which was less than 0.05.Hence this

observation was very significant.

56

Similarly the findings in frozen section study (B) were correlated with

histopathological examination(C). The results were tabulated.

57

Table -11 Correlation between Frozen Section study (B) and Histopathological Examination (C)

Histopathology (C)

To

tal

Lo

w g

rade

glio

ma

Hig

h g

rad

e

glio

ma

Sm

all

rou

nd

ce

ll tu

mo

ur

Me

nin

gio

ma

Sch

wan

no

ma

Pitu

ita

ry

Ad

en

om

a

Me

tasta

tic

Carc

ino

mato

u

s D

ep

osits

Hem

an

gio

bla

sto

ma

Ep

ide

rmo

id

Cyst

Gra

nu

lom

ato

us L

esio

n

Ma

lign

an

t

Me

lan

om

a

Ep

en

dym

om

a

Frozen Section

Study(B)

Low grade glioma Count 8 2 2 0 0 0 0 0 0 0 0 0 12

% within Frozen Section

Study(B)

66.7% 16.7% 16.7% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

High grade glioma Count 1 8 0 0 0 0 0 0 0 0 0 0 9

% within Frozen Section

Study(B)

11.1% 88.9% .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Small round cell tumour Count 1 0 3 0 0 0 0 0 0 0 0 0 4

% within Frozen Section

Study(B)

25.0% .0% 75.0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Meningioma Count 0 0 0 6 0 0 0 0 0 0 0 0 6

% within Frozen Section

Study(B)

.0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Schwannoma Count 0 0 0 0 3 0 0 0 0 0 0 0 3

% within Frozen Section

Study(B)

.0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% .0% 100.0%

Pituitary Adenoma Count 0 0 0 0 0 1 0 0 0 0 0 0 1

58

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% .0% 100.0%

Metastatic Carcinomatous

Deposits

Count 0 0 0 0 0 0 3 0 0 0 0 0 3

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% .0% 100.0%

Hemangioblastoma Count 0 0 0 0 0 0 0 1 0 0 0 0 1

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% .0% 100.0%

Epidermoid Cyst Count 0 0 0 0 0 0 0 0 2 0 0 0 2

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% .0% 100.0%

Granulomatous Lesion Count 0 0 0 0 0 0 0 0 0 3 0 0 3

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% .0% 100.0%

Malignant Melanoma Count 0 0 0 0 0 0 0 0 0 0 1 0 1

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% .0% 100.0%

Ependymoma Count 0 0 0 0 0 0 0 0 0 0 0 5 5

% within Frozen Section

Study(B)

.0% .0% .0% .0% .0% .0% .0% .0% .0% .0% .0% 100.0% 100.0%

Total Count 10 10 5 6 3 1 3 1 2 3 1 5 50

% within Frozen Section

Study(B)

20.0% 20.0% 10.0% 12.0% 6.0% 2.0% 6.0% 2.0% 4.0% 6.0% 2.0% 10.0% 100.0%

59

The above table correlates the finding in frozen section study with

histopathological examination. There was one hundred percent correlation in the case

of meningioma, schwannoma, hemangioblastoma, ependymoma, pituitary adenoma,

metastatic carcinomatous deposits, epidermoid cyst and granulomatous lesions. In the

case of low grade glioma it was 66.7% and in high grade glioma it was 88.9%.

66.7

88.9

75

100 100 100 100 100 100 100 100 100 100

0

20

40

60

80

100

120

Correlation between Frozen Section Study and Histopathology

60

Chi-Square Tests

Value df

Asymp. Sig. (2-

sided)

Pearson Chi-Square 491.528a 121 .000

Likelihood Ratio 191.262 121 .000

Linear-by-Linear Association 48.067 1 .000

N of Valid Cases 50

The p value was 0.000 which was less than 0.05 hence this

observation was very significant.

COLOUR PLATES

Squash Cytology showing sheets of reactive astrocytes exhibiting moderate

nuclear pleomorphism in a fibrillary background

Squash smear cytology showing sheets of neoplastic astrocytes, moderate

nuclear atypia

FIG 1:GLIOMA

GGLIOGLIOM

A

FIG 2: HIGH GRADE GLIOMA

Squash smear cytology showing sheets of neoplastic astrocytes with minimal

atypia traversed by endothelium lined blood vessels

Squash smear cytology showing whorls of meningothelial cells with

moderate eosinophilic cytoplasm and ovoid bland nucleus

FIG 4: MENINGIOMA

FIG 3: LOW GRADE

GLIOMA

Squash smear cytology of pituitary adenoma on low power showing a

cellular smear with cohesive clusters on acinar configuration and singly

dispersed

Pituitary adenoma on high power showing cells with scant cytoplasm and round uniform nucleus

FIG 5: PITUITARY ADENOMA- LOW POWER

FIG 6: PITUITARY ADENOMA-HIGH POWER

Squash smear cytology of Schwannoma in low power showing sheets of

spindle shaped cells

Schwannoma in squash smear cytology on higher power showing sheets of

spindle shaped cells with spindle shaped nuclei

FIG 7: SCHWANNOMA-LOW POWER

FIG 8: SCHWANNOMA-HIGH POWER

Meningioma in frozen section showing fascicles and whorls of meningothelial cells

Meningioma in frozen section on higher power showing intranuclear pseudo inclusions

FIG 9: MENINGIOMA IN FROZEN SECTION

FIG 10: MENINGIOMA –HIGH POWER

Schwannoma in frozen section on low power showing spindle shaped cells

with hyper cellular and hypo cellular areas

FIG 11: MENINGIOMA IN FROZEN SECTION SHOWING PSAMMOMA BODIES

FIG 12: SCHWANNOMA IN FROZEN SECTION

Schwannoma in frozen section showing hyper cellular areas with nuclear

palisading and verocay bodies and freezing artefact

Low grade glioma on frozen section showing mild hyper cellularity and uneven

distribution of atypical cells

FIG 13 : FROZEN SECTION-SCHWANNOMA WITH FREEZING ARTEFACT

FIG 14: LOW GRADE GLIOMA ON FROZEN SECTION

Caseating granulomatous lesion on frozen section demonstrating multiple epithelioid granulomata

Frozen section of malignant melanoma showing cells with intracytoplasmic

dense brown melanin pigmentation and pleomorphic hyperchromatic nuclei

FIG 15: CASEATING GRANULOMATOUS LESION ON FROZEN SECTION

FIG 16: MALIGNANT MELANOMA ON FROZEN SECTION

Pilocytic Astrocytoma demonstrating sheets of polyhedral cells and numerous microcystic spaces

FIG 17 A & 17 B: PILOCYTIC ASTROCYTOMA ON HISTOPATHOLOGY

Glioblastoma on HPE showing pleomorphic astrocytes and areas of necrosis

Glioblastoma in HPE showing sheets of neoplastic astrocytes with

pleomorphic hyperchromatic nuclei and proliferating blood vessels

FIG 18: HISTOPATHOLOGICAL EXAMINATION OF

GLIOBLASTOMA ON LOW POWER

FIG 19: GLIOBLASTOMA ON HIGHER POWER

Schwannoma on low power showing a neoplasm arranged in fascicles

composed of spindle shaped cells with nuclear palisading and verocay

bodies

Pituitary adenoma on HPE showing sheets of uniform cells with scant

cytoplasm and round uniform nucleus

FIG 20: SCHWANNOMA ON HPE

FIG 21: PITUITARY ADENOMA

Meningioma on HPE showing plump polyhedral cells with eosinophilic

cytoplasm and uniform vesicular nuclei

Neoplasm arranged in cords, sheets and perivascular pseudo rosettes

FIG 22: MENINGIOMA ON HPE

FIG 23: EPENDYMOMA ON HPE

DISCUSSION

61

DISCUSSION

Intraoperative diagnosis is essential for the operating

neurosurgeon in order to delineate the neoplastic and non-

neoplastic lesions, to identify the treatable inflammatory

conditions and to know whether a lesion is primary pathology of

CNS or secondary affliction from other sites by which one can

proceed accordingly. Even though histopathology is considered

‘gold standard’ it cannot be done immediately to give a rapid

diagnosis.

Frozen section was introduced some hundred years ago but

it has its own limitations in CNS as shown by Folkerth et al 19

and Shah et al 14. Squash smear cytology is known to give rapid

results 40 but it has to compare positively with histopathology to

be considered useful.

There are certain studies which highlighted the advantages

and disadvantages of these two procedures i.e. frozen section

study and squash smear study 8, 11, 41. In general frozen section

was having good correlation with histopathology in firm

tumours like meningioma and schwannoma when giving

accurate architectural details. It was not useful in soft lesions

since it needed more tissue and it resulted in freezing artefact 42,

43. On the other hand squash smear cytology showed better

details regarding type of background and specific cellular

elements. It also needed tiny tissue sample which would be the

case in most of the surgical procedures.

62

Savargaonkar et al 4 in the year 2001 published the pros and

cons of these two procedures and concluded that cytology

technique was more useful for astrocytoma, small round cell

tumours and certain metastases. The frozen section technique

was better for the diagnosis of meningioma, reactive lesions,

ependymomas and most metastatic lesions.

We had undertaken this study in order to find the usefulness

of squash smear cytology and frozen section study in the

intraoperative diagnosis of CNS lesions by comparing among

them and with histopathology.

We have used the techniques of squash smear cytology,

frozen section study followed by histopathological examination

in 50 patients for whom neurosurgical procedures were

contemplated in our institution and analysed the results.

Out of 50 patients there were 26 male patients and 24

female patients. The median age was 42. We had varied

diagnosis among our group of patients. As many as nineteen

patients were diagnosed to have gliomas and seven patients were

having meningioma when wheeled in for operative procedure.

In our study 40 percent of the lesions were present in the

frontal lobe mostly gliomas which was comparable with the

current incidence10. We had 45 neoplastic lesions and only 5 non

neoplastic lesions among the total 50 patients (90%) which was

the case in other such studies also 1, 8 .Among the neoplastic

lesions 20 patients were having gliomas which included two

63

cases of Pilocytic astrocytoma, one case of diffuse astrocytoma,

one case of mixed astrocytoma, two cases of anaplastic

astrocytoma, one case of Oligodendroglioma, one case of

anaplastic Oligodendroglioma and seven cases of glioblastoma

multiforme.

There were six cases of meningioma which included two

cases of meningothelial meningioma and one case of atypical

meningioma (WHO grade II). There were three cases of

schwannoma, one Medulloblastoma and one atypical teratoid

rhabdoid tumour. There was one pituitary adenoma, six cases of

ependymoma, two cases of esthesioneuroblastoma (growth

nasopharynx and nasal cavity with intracranial extension) and

three metastatic carcinomatous deposits.

Though ependymomas are gliomas they were considered

separately as their cytology was typical. Of the five non-

neoplastic lesions two were epidermoid cysts, two cases were

caseating granulomatous lesion and one was fungal granuloma.

The findings of squash smear cytology, frozen section study

and histopathology were compared and analysed. In 82% of the

patients all the three showed the same diagnosis. In one patient

Squash smear cytology and histopathology were same but

frozen section study was different. In total 84% of the patients

were accurately diagnosed by squash smear cytology alone

intraoperatively. Similar observations were made by Vikram

Nanamg et al 1 who studied 75 patients. They had concluded that

squash smear study had a diagnostic accuracy of 89.2%.

64

Hephzibah Rani et al 8 studied 110 cases and concluded that the

diagnosis on squash smear study correlated with final diagnosis

in 103 cases.

The 42 patients (84%) in whom squash smear cytology alone

would have fetched correct intraoperative diagnosis included

low grade and high grade gliomas that could easily been

diagnosed because of their fibrillary background, ependymomas,

granulomatous lesions, metastatic carcinomatous deposits,

medulloblastomas, meningioma, schwannoma and

esthesioneuroblastoma.

In one patient squash smear study showed high grade glioma

which was seen as low grade glioma in frozen section study and

histopathological examination revealed small round cell tumour

(lymphoma). We did immunohistochemistry to get final

diagnosis and it was found out to be metastatic small cell

carcinomatous deposit probably from lung.

In another patient squash smear study and frozen section

study showed metastatic carcinomatous deposits but in

histopathology the possibility of metastatic poorly differentiated

carcinoma and high grade glioma were considered. In this

patient immunohistochemistry was done where GFAP was

strongly positive and CK was negative. Hence a diagnosis of

high grade glioma was established.

The diagnostic accuracy of squash smear study and frozen

section study were analysed.

65

Table 12- Diagnostic accuracy of Squash smear Cytology

Diagnosis by Squash

Smear Cytology

Misdiagnosed

Cases

Correctly

Diagnosed

Diagnostic

Accuracy

Gliomas (19) 2 17 89.5%

Small round cell

tumour(5)

2 3 60%

Meningioma (6) Nil 6 100%

Schwannoma (3) Nil 3 100%

Pituitary adenoma (1) Nil 1 100%

Metastatic

carcinomatous

deposits(3)

Nil 3 100%

Hemangioblastoma (1) Nil 1 100%

Ependymoma (5) Nil 5 100%

Malignant Melanoma

(1)

Nil 1 100%

Epidermoid Cyst(2) Nil 2 100%

Granulomatous Lesion

(3)

Nil 3 100%

Organised Hematoma(1) 1 -

66

Glioma:

In the present study 19 cases of glioma were diagnosed by

squash smear cytology. On the other hand frozen section study

showed 21 cases of glioma. Of these 19 cases by squash smear,

grading was accurately done in 14 cases with squash smear

cytology alone.

In histopathology these gliomas were found to be

astrocytoma and oligodendrogliomas. In three cases glioma was

found to be high grade – glioblastoma, NOS, (WHO grade IV),

radiologically showed large contrast enhancing ring pattern

surrounded by peritumoural oedema creating mass effect,

anaplastic astrocytoma (WHO Grade III), and anaplastic

oligodendroglioma (WHO Grade III) - by histopathology. The

diagnosis of glioblastoma NOS is reserved for those tumours for

which full IDH evaluation cannot be performed.

Diagnostic accuracy without grading was 89.5% but with

grading it was 74%. In two cases a diagnosis of small round cell

tumour was made by histopathology. In one of these cases,

immunohistochemistry revealed metastatic small cell carcinoma

probably from lung. In this particular patient squash smear

cytology showed cellular smears with sheets , clusters and

singly dispersed polyhedral cells with dark staining nuclei and

frozen section showed low grade glioma but histopathology

section showed neoplasm composed of sheets of small round

cells with scant cytoplasm and dark staining nuclei and a

67

possibility of small round cell tumour(lymphoma) was

considered.

Using frozen section study diagnostic accuracy with grading

i.e. low grade and high grade was 76.2%. The main drawback in

frozen section was presence of freezing artefact.

The clear distinction between low grade glioma and high

grade glioma was adequate. It is not essential to distinguish

between an oligo dendroglioma and an astrocytoma

intraoperatively as it will not affect the surgical contemplation36.

68

Small round cell tumour:

In the present study three cases of medulloblastomas and

two cases of olfactory neuroblastoma were diagnosed by squash

smear cytology. Of the three cases of medulloblastomas, frozen

section gave the same impression in one patient and in other

patient a diagnosis of ependymoma was made. In the first

instance histopathology gave a diagnosis of mixed oligo

astrocytoma (WHO grade II) and in the second the diagnosis of

ependymoma was confirmed.

Meningioma:

There were six cases of meningioma out of which five were

meningothelial meningioma (WHO grade I) and one was

atypical meningioma (WHO grade II). There was 100%

diagnostic accuracy in squash smear cytology as well as frozen

section study.

Using squash smear cytology meningioma showed cellular

smears composed of plump elongated cells arranged in sheaths

and whorls with round to ovoid nuclei with some showing intra

nuclear pseudo inclusions.

Frozen section and histopathology showed neoplasm

arranged as whorls, fascicles and foci of Psammoma bodies. In

one patient a diagnosis of atypical meningioma was made by all

the three techniques.

69

Schwannoma:

There was 100% diagnostic accuracy in squash smear

cytology as well as frozen section study. There were three cases

of schwannoma. In squash cytology, smears were cellular

showing cohesive clusters of spindle shaped cells admixed with

a few loosely cohesive cells in a haemorrhagic background.

In frozen section study and histopathology, section showed

a neoplasm with hyper cellular and hypo cellular areas

composed of spindle shaped cells with elongated nucleus. Hyper

cellular areas showed verocay bodies.

Pituitary adenoma:

There was a case of pituitary adenoma with 100 % diagnostic

accuracy. Squash smear cytology showed cellular smears with

cohesive clusters, acinar configuration and singly dispersed

fairly uniform cells with scant cytoplasm and round uniform

nucleus in a haemorrhagic background.

Frozen section and histopathology showed a neoplasm

composed of uniform cells arranged in nests, trabeculae and

sheets with scant cytoplasm and round uniform nucleus.

Metastasis:

There were three cases of metastatic carcinomatous deposits

diagnosed in squash which was the same in the frozen section

but one case turned out to be high grade glioma in

immunohistochemistry.

70

Hemangioblastoma:

There was one case of hemangioblastoma in a 45 year old

male with multiple cerebellar space occupying lesions. Squash

smear cytology showed moderately cellular smears with sheets

and clusters of round to oval cells with dark staining nuclei and

scant eosinophilic cytoplasm admixed with large polyhedral

cells with peripherally pushed dark staining nuclei interspersed

with numerous thin walled vascular channels. Frozen section

and HPE revealed same diagnosis.

Granulomatous lesions:

In squash, two cases of granulomatous lesions probably

tuberculosis was given which showed lymphocytes, histiocytes

and occasional epithelioid cells in a serofibrinous background.

Frozen section showed many areas with freezing artefact

and ill formed epithelioid granulomata and caseous necrosis.

Histopathological examination revealed caseating

granulomatous lesion.

There was a fungal granuloma diagnosed in squash which

showed septate fungal hyphae with epithelioid histiocytes, ill

formed granulomata in a necrotic background. In frozen section,

multiple epithelioid granuloma with scattered fungal hyphae was

seen and histopathology revealed fungal granuloma.

71

Epidermoid Cysts:

There were two cases of epidermoid cysts diagnosed in this

study with 100% accuracy. Epidermoid cyst may be confused

with craniopharyngeoma but characteristic cytology features and

clinico radiological data can help in the differentiation44,45,46

Malignant melanoma:

One case of malignant melanoma was given with squash and

frozen section in a 55 year old female with skull base SOL.

Intraoperatively it was a blackish mass which was firm, friable

and vascular. In Squash it was a moderately cellular smear

showing sheets, clusters and singly dispersed cells with

pleomorphic hyperchromatic nuclei many exhibiting prominent

nucleoli. Many cells showed brown pigmentation in the

cytoplasm. Histopathology confirmed the diagnosis.

Ependymoma:

There were five cases of Ependymoma diagnosed with

squash smear cytology with 100% diagnostic accuracy.

Smears showed clusters of round to oval cells with

moderate eosinophilic cytoplasm and dark staining nuclei. Many

foci showed perivascular pseudorosettes. Background was

fibrillary.

Frozen section showed neoplasm arranged in sheets with

perivascular pseudorosettes. But many areas of freezing artefact

were present.

72

Sensitivity and specificity of squash smear cytology:

The sensitivity and specificity of squash smear cytology was

computed by chi square test. They were 83.3% and 100%

respectively with p value 0.004 which is less than 0.05 hence

very significant. The positive predictive value was 100%.

FIG 24: ROC CURVE SHOWING SENSITIVITY AND SPECIFICITY

73

Table 13: Diagnostic accuracy of squash Smear cytology –

Comparison of various studies

S .No Name of the study Diagnostic Accuracy

1. Martinez et al17 70%

2. Kalpana et al47 91.1%

3. Imitiaz et al48 84%

4. Shah AB et al14 89.7%

5. Sumit et al34 88.5%

6. In this study 85.6%

The above observation suggests that squash smear cytology

has a acceptable sensitivity and high specificity with good

diagnostic accuracy. Hence it can be used as a rapid intra

operative diagnostic tool in CNS lesions to assist the operating

surgeon in his or her decision making.

However it cannot be used as a sole diagnostic test but to

be confirmed by histopathology. On the other hand frozen

section study needed high cost equipment, time, expertise and

sizeable tissue sample which would not be available in all the

cases and at the same time it was compounded by freezing

artefact.

74

SUMMARY

Intraoperative diagnosis of CNS lesions is very important to the operating

neurosurgeon. The frozen section study was used for the past 100 years but with

limitations. Squash smear cytology is rapid, reliable and less time consuming as

suggested by many studies. The present study analysed the accuracy of squash

smear cytology in rapid diagnosis of CNS lesions intra operatively in relation to

frozen section study. Fifty patients presenting to neurosurgery were included in

this study. Squash smear cytology, frozen section study and histopathology

examination were done in all these cases and results analysed.

The following observations were made.

Male female ratio was 1.08:1

Median age was 42 years

Most common clinico radiological diagnosis was glioma

Frontal lobe of brain was commonly involved.

There were 45 cases of neoplastic and 5 cases of non -neoplastic cases.

Most common tumour was glioma followed by meningioma.

Squash smear cytology had a diagnostic accuracy of 85.6% which was similar

to previous studies.

Frozen section study was limited by freezing artefact, equipment cost, time and

expertise and it had no unblemished upper hand over squash smear cytology.

Sensitivity of squash smear cytology was 83.3% and its specificity was 100%

with positive predictive value of 100%.

SUMMARY

SUMMARY

75

CONCLUSION

Squash smear cytology is a sensitive and specific modality

for diagnosing CNS lesions. The method is easy, rapid,

inexpensive and requires only tiny tissue sample. The details of

cellular morphology are well seen in squash smear cytology.

Hence squash cytology can be used as a reliable diagnostic tool

in developing countries like India since the cost of cryostat is

prohibitive and unlike cryostat squash cytology does not require

electricity for slide preparation. Moreover cryostat needs

maintenance and an experienced microtomist to cut the section

and the problems of freezing artefact cause diagnostic pitfalls.

However squash smear cytology should not be used as a sole

diagnostic tool but to be confirmed with histopathology which is

‘gold standard’.

With increasing use of stereotactic biopsies squash cytology

provides means of assessing the tissue adequacy and also

provides rapid diagnosis. In the hands of experienced

pathologist with good exposure to neuropathology squash

cytology is an accurate and reliable procedure for rapid

diagnosis of CNS lesions.

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ANNEXURE I

WHO CLASSIFICATION AND HISTOLOGICAL GRADING OF CNS

TUMOURS(2016) 37

ANNEXURES

ANNEXURE II

PROFORMA

INSTITUTE OF PATHOLOGY,

MADRAS MEDICALCOLLEGE &

RAJIV GANDHI GOVERNMENT GENERAL HOSPITAL

CHENNAI-600 003. TAMILNADU. INDIA.

Name:

Age/Sex:

IP No:

Income:

Occupation:

Study Number:

History of Present Illness:

Past Medical & Surgical History:

Family History:

Basic Investigations:

Computerised Tomography:

Magnetic Resonance Imaging:

Intraoperative Findings:

Squash Smear Examination:

Frozen Section Examination:

Histopathological Examination:

Immunohistochemistry (If Needed):

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sis

Sq

ua

sh D

iag

no

sis

Fro

zen

Sec

tio

n

Dia

gn

osi

s H

PE

Dia

gn

osi

s IH

C i

f

don

e

1.

48

F

Men

ingio

ma

Men

ingio

ma

Men

ingio

ma

Men

ingio

ma

-

2.

55

F

? H

igh G

rad

e G

lio

ma

? M

etas

tasi

s

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Gli

ob

last

om

a -

3.

40

F

Gli

om

a

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

-

4.

62

M

Hig

h G

rad

e N

eop

lasm

H

igh

Gra

de

Gli

om

a

Hig

h G

rad

e G

lio

ma

Gli

ob

last

om

a -

5.

55

M

Sch

wan

no

ma

Sch

wan

no

ma

Sch

wan

no

ma

Sch

wan

no

ma

-

6.

55

F

Hig

h G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

-

7.

50

F

Pit

uit

ary M

acro

aden

om

a

Pit

uit

ary a

den

om

a

Pit

uit

ary a

den

om

a

Pit

uit

ary a

den

om

a

-

8.

8

M

- S

mal

l R

ou

nd

Cel

l

Tu

mo

ur

Med

ull

obla

sto

ma

Mix

ed

Oli

go

astr

ocyto

ma

-

9.

42

M

Sch

wan

no

ma

S

chw

ann

om

a

Sch

wan

no

ma

Sch

wan

no

ma

-

10

. 6

0

M

Epid

erm

oid

Cyst

E

pid

erm

oid

Cyst

E

pid

erm

oid

Cyst

E

pid

erm

oid

Cyst

-

11

. 5

2

F

Gli

ob

last

om

a

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Pil

ocyti

c A

stro

cyto

ma

-

12

. 4

2

F

Men

ingio

ma

Men

ingio

ma

M

enin

gio

ma

M

enin

gio

ma

13

. 2

6

M

Ara

chn

oid

Cyst

E

pid

erm

oid

Cyst

E

pid

erm

oid

Cyst

E

pid

erm

oid

Cyst

14

. 3

7

F

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Sm

all

Ro

und C

ell

Tu

mour

Met

asta

tic

Sm

all

Cel

l C

a

pro

bab

ly

from

lung

15

. 4

2

M

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Gli

ob

last

om

a -

16

. 2

3

M

Oli

go

den

dro

gli

om

a

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Dif

fuse

Oli

go

den

dro

gli

om

a

-

17

. 1

5

F

? E

pen

dym

om

a

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Pil

ocyti

c A

stro

cyto

ma

-

18

. 4

2

M

Men

ingio

ma

Men

ingio

ma

M

enin

gio

ma

M

enin

gio

ma

-

19

. 2

M

Med

ull

obla

sto

ma/

Aty

pic

al

Ter

ato

id R

hab

do

id

Tu

mo

ur

Med

ull

obla

sto

ma

Ep

end

ym

om

a

Epen

dym

om

a

-

20

. 6

0

M

Hig

h G

rad

e

Gli

om

a/M

etas

tasi

s

Met

asta

tic

Les

ion

M

etas

tati

c

Car

cino

mat

ous

Dep

osi

ts

1.M

etas

tati

c P

oorl

y

Dif

fere

nti

ated

Ca

2. H

igh G

rade

Gli

om

a

-

21

. 1

0

M

Epen

dym

om

a

Ep

end

ym

om

a

Ep

end

ym

om

a

Epen

dym

om

a

-

22

. 4

8

F

? S

econ

dar

ies

?Tuber

culo

ma

Met

asta

tic

Car

cin

om

ato

us

Dep

osi

ts

Met

asta

tic

Car

cino

mat

ous

Dep

osi

ts

Met

asta

tic

Ad

eno

carc

ino

mat

ou

s

Dep

osi

ts

-

23

. 2

5

F

Hig

h G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Gli

ob

last

om

a -

24

. 4

4

F

Sch

wan

no

ma

Ep

end

ym

om

a

Ep

end

ym

om

a

Epen

dym

om

a

-

25

. 5

5

F

? M

elan

om

a

Mal

ignan

t M

elan

om

a

Mal

ignan

t M

elan

om

a

Mal

ignan

t M

elan

om

a

-

26

. 5

8

M

Hig

h G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

An

apla

stic

Ast

rocyto

ma

-

27

. 1

6

F

Epen

dym

om

a/A

typ

ical

Men

ingio

ma

Ep

end

ym

om

a

Ep

end

ym

om

a

Epen

dym

om

a

-

28

. 4

3

M

Est

hes

ion

euro

bla

sto

ma

Sm

all

Ro

un

d C

ell

Tu

mo

ur

Sm

all

Ro

und C

ell

Tu

mour

Est

hes

ion

euro

bla

sto

ma

-

29

. 1

3

M

Oli

go

den

dro

gli

om

a

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

An

apla

stic

Oli

go

den

dro

gli

om

a

-

30

. 3

1

M

? G

lio

ma

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

-

31

. 4

1

M

Gra

nulo

mat

ou

s L

esio

n ?

TB

Gra

nu

lom

ato

us

Les

ion

Pro

bab

ly T

B

Cas

eati

ng

Gra

nu

lom

atous

Les

ion

Cas

eati

ng

Gra

nulo

mat

ous

Les

ion

-

32

. 4

6

M

Ast

rocyto

ma

Lo

w G

rad

e G

lio

ma

Lo

w G

rad

e G

lio

ma

Gan

gli

og

lio

ma

-

33

. 3

5

F

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Hig

h G

rad

e G

lio

ma

Gli

ob

last

om

a -

34

. ½

M

A

typic

al T

erat

oid

Rhab

doid

Tu

mo

ur

Ep

end

ym

om

a

Ep

end

ym

om

a

Aty

pic

al T

erat

oid

Rhab

doid

Tu

mour

-

35

. 7

F

? M

edull

ob

last

om

a

? A

typic

al T

erat

oid

Rhab

doid

Tu

mo

ur

Med

ull

obla

sto

ma

M

edull

obla

sto

ma

M

edull

obla

sto

ma

-

36

. 2

8

F

? L

ow

Gra

de

Gli

om

a

Lo

w G

rad

e G

lio

ma

L

ow

Gra

de

Gli

om

a

Dif

fuse

Ast

rocyto

ma

-

37

. 2

3

M

Sch

wan

no

ma

S

chw

ann

om

a

Sch

wan

no

ma

Sch

wan

no

ma

-

38

. 2

1

M

Gli

om

a

Lo

w G

rad

e G

lio

ma

L

ow

Gra

de

Gli

om

a

Oli

go

den

dro

gli

om

a

-

39

. 7

0

F

Cer

ebel

lar

Met

asta

sis

Met

asta

tic

Car

cin

om

ato

us

Dep

osi

ts

Met

asta

tic

Car

cino

mat

ous

Dep

osi

ts

Met

asta

tic

Car

cino

mat

ous

Dep

osi

ts f

rom

infi

ltra

ting d

uct

al C

a

-

Bre

ast

40

. 3

7

F

Epen

dym

om

a

Ep

end

ym

om

a

Ep

end

ym

om

a

Epen

dym

om

a

-

41

. 1

8

F

Tu

ber

culo

ma

Cas

eati

ng

Gra

nu

lom

ato

us

Les

ion

Cas

eati

ng

Gra

nu

lom

atous

Les

ion

Cas

eati

ng

Gra

nulo

mat

ous

Les

ion

-

42

. 6

7

M

Men

ingio

ma

M

enin

gio

ma

M

enin

gio

ma

M

enin

gio

ma

-

43

. 3

8

F

Men

ingio

ma

M

enin

gio

ma

M

enin

gio

ma

M

enin

gio

ma

-

44

. 4

5

F

Gli

ob

last

om

a

Hig

h G

rad

e G

lio

ma

H

igh

Gra

de

Gli

om

a

Gli

ob

last

om

a -

45

. 4

5

F

Hem

angio

bla

sto

ma

Hem

ang

iob

last

om

a

Hem

angio

bla

sto

ma

Hem

angio

bla

sto

ma

-

46

. 9

F

Gli

om

a

Ep

end

ym

om

a

Ep

end

ym

om

a

Epen

dym

om

a

-

47

. 3

8

M

Ab

sces

s F

un

gal

Les

ion

F

un

gal

Gra

nulo

ma

F

ungal

Gra

nulo

ma

-

48

. 6

2

M

? H

igh G

rad

e G

lio

ma

? C

alci

fied

Blo

od

Clo

t

Org

anis

ed H

emat

om

a

Hig

h G

rad

e G

lio

ma

An

apla

stic

Ast

rocyto

ma

-

49

. 4

9

F

? A

typic

al M

enin

gio

ma

Aty

pic

al M

enin

gio

ma

A

typ

ical

Men

ingio

ma

A

typic

al M

enin

gio

ma

-

50

. 4

7

M

Est

hes

ion

euro

bla

sto

ma

E

sth

esio

neu

rob

last

om

a

Est

hes

ion

euro

bla

sto

ma

E

sth

esio

neu

rob

last

om

a

-

ANNEXURE IV -MASTER CHART II-CORRELATION

S. N0

Sex Age

Group Provisional Diagnosis

Site Of Lesion

Neoplastic Squash Smear

Cytology(A)

Frozen Section

Study(B)

Histopathology (C)

Correlation between A,B & C

1. 2 7 4 1 1 4 4 4 1

2. 2 8 2 1 1 2 2 2 1

3. 2 6 4 1 1 1 1 1 1

4. 1 9 2 7 1 2 2 2 1

5. 1 8 5 4 1 5 5 5 1

6. 2 8 2 6 1 1 2 1 2

7. 2 7 6 5 1 6 6 6 1

8. 1 3 3 8 1 3 3 1 4

9. 1 7 5 4 1 5 5 5 1

10. 1 8 9 2 2 9 9 9 1

11. 2 8 2 3 1 1 1 1 1

12. 2 7 4 1 1 4 4 4 1

13. 1 5 0 1 2 9 9 9 1

14. 2 6 2 1 1 2 1 3 5

15. 1 7 2 1 1 2 2 2 1

16. 1 5 1 1 1 1 1 1 1

17. 2 4 11 9 1 13 13 13 1

18. 1 7 4 1 1 4 4 4 1

19. 1 2 3 9 1 3 1 1 3

20. 1 8 7 3 1 7 7 7 1

21. 1 3 11 1 1 13 13 13 1

22. 2 7 7 1 1 7 7 7 1

23. 2 5 2 12 1 1 2 2 3

24. 2 7 5 11 1 13 13 13 1

25. 2 8 0 1 1 11 11 11 1

26. 1 8 2 10 1 1 1 2 4

27. 2 4 1 1 1 1 1 1 1

28. 1 7 3 1 1 3 3 3 1

29. 1 4 1 3 1 1 1 2 4

30. 1 6 1 8 1 2 2 2 1

31. 1 7 10 9 2 10 10 10 1

32. 1 7 1 10 1 1 1 1 1

33. 2 6 2 1 1 2 2 2 1

34. 1 1 0 10 1 1 1 3 4

35. 2 3 3 8 1 3 3 3 1

36. 2 5 1 2 1 1 1 1 1

37. 1 5 5 4 1 5 5 5 1

38. 1 5 1 7 1 1 1 1 1

39. 2 9 7 9 1 7 7 7 1

40. 2 6 11 10 1 13 13 13 1

41. 2 4 10 2 2 10 10 10 1

42. 1 9 4 1 1 4 4 4 1

43. 2 6 4 1 1 4 4 4 1

44. 2 7 2 7 1 2 2 2 1

45. 1 7 8 9 1 8 8 8 1

46. 2 3 1 9 1 13 13 13 1

47. 1 6 10 2 2 10 10 10 1

48. 1 9 2 1 1 12 2 2 3

49. 2 7 4 1 1 4 4 4 1

50. 1 7 3 1 1 3 3 3 1

ANNEXURE V - KEY TO MASTER CHART

Sex: Male (1) Female (2)

Age group

Less than 1 year (1)

1 -5 yrs (2)

6-10 yrs (3)

11-20 yrs (4)

21-30 yrs (5)

31-40 yrs (6)

41-50 yrs (7)

51-60 yrs (8)

More than 60 yrs (9)

Provisional diagnosis

Low grade glioma (1)

High grade glioma (2)

Small round cell tumour (3)

Meningioma (4)

Schwannoma (5)

Pituitary Adenoma (6)

Metastasis (7)

Hemangioblastoma (8)

Epidermoid Cyst (9)

Granulomatous Lesion (10)

Ependymoma (11)

Others (0)

Site of lesion

Frontal (1)

Temporal (2)

Parietal (3)

Cerebello Pontine Angle (4)

Sellar/Suprasellar (5)

Thalamus (6)

Corpus Callosum (7)

Brain Stem (8)

Cerebellum (9)

Ventricle (10)

Base Of Skull (11)

Spine (12)

Squash Smear Cytology (A)

Low grade glioma (1)

High Grade Glioma (2)

Small round Cell tumour (3)

Meningioma (4)

Schwannoma (5)

Pituitary Adenoma (6)

Metastatic Carcinomatous Deposits (7)

Hemangioblastoma (8)

Epidermoid Cyst (9)

Granulomatous Lesion (10)

Malignant Melanoma (11)

Organised Hematoma (12)

Ependymoma (13)

Frozen section study (B)

Low grade glioma (1)

High Grade Glioma (2)

Small round Cell tumour (3)

Meningioma (4)

Schwannoma (5)

Pituitary Adenoma (6)

Metastatic Carcinomatous Deposits (7)

Hemangioblastoma (8)

Epidermoid Cyst (9)

Granulomatous Lesion (10)

Malignant Melanoma (11)

Organised Hematoma (12)

Ependymoma (13)

Histopathology (C)

Low grade glioma (1)

High Grade Glioma (2)

Small round Cell tumour (3)

Meningioma (4)

Schwannoma (5)

Pituitary Adenoma (6)

Metastatic Carcinomatous Deposits (7)

Hemangioblastoma (8)

Epidermoid Cyst (9)

Granulomatous Lesion (10)

Malignant Melanoma (11)

Organised Hematoma (12)

Ependymoma (13)

Correlation Between A,B & C

A, B & C reveal same diagnosis (1)

A & C reveal same diagnosis but B is different (2)

B & C reveal same diagnosis but A is different (3)

A & B reveal same diagnosis but C is different (4)

A , B & C are all different (5)

ANNEXURE VI

INFORMATION SHEET

We are conducting a study on comparison of squash and frozen section with

final HPE diagnosis in CNS lesions among patients attending Government

General Hospital, Chennai and for that your specimen may be valuable to us.

The purpose of this study is to compare the diagnostic accuracy of squash and

frozen section in CNS lesions.

We are selecting CNS lesions requested for intra-operative diagnosis for both

squash and frozen section which in any way do not affect your final report or

management.

The privacy of the patients in the research will be maintained throughout the

study. In the event of any publication or presentation resulting from the

research, no personally identifiable information will be shared.

Taking part in this study is voluntary. You are free to decide whether to

participate in this study or to withdraw at any time; your decision will not

result in any loss of benefits to which you are otherwise entitled.

The results of the special study may be intimated to you at the end of the study

period or during the study if anything is found abnormal which may aid in the

management or treatment.

Signature of investigator Signature of participant

Date:

ANNEXURE VII -INFORMED CONSENT FORM

Title of the study:

A comparative study of squash, Frozen section and Histopathological examination in the

diagnosis of Central nervous system Lesions

Name of the Participant :

Name of the Principal (Co-Investigator) :

Name of the Institution : Madras Medical College

Name and address of the sponsor / agency (ies) (if any) :

Documentation of the informed consent

I _____________________________ have read the information in this form (or it has been

read to me). I was free to ask any questions and they have been answered. I am over 18 years

of age and, exercising my free power of choice, hereby give my consent to be included as a

participant in “A comparative study of squash, Frozensection and Histopathological

examination in the diagnosis of central nervous system lesions”.

1. I have read and understood this consent form and the information provided to me.

2. I have had the consent document explained to me.

3. I have been explained about the nature of the study in which the operative CNS lesions will

be subjected to squash, frozen section and histopathological examination and immuno-

histochemistry if necessary.

4. I have been explained about my rights and responsibilities by the investigator. I have the right

to withdraw from the study at any time.

5. I have been informed the investigator of all the treatments I am taking or have taken in the

past ________ months including any native (alternative) treatment.

6. I hereby give permission to the investigators to release the information obtained from me as

result of participation in this study to the sponsors, regulatory authorities, Govt. agencies, and

IEC. I understand that they are publicly presented.

7. I have understood that my identity will be kept confidential if my data are publicly presented

8. I have had my questions answered to my satisfaction.

9. I have decided to be in the research study.

I am aware that if I have any question during this study, I should contact the investigator. By

signing this consent form I attest that the information given in this document has been clearly

explained to me and understood by me, I will be given a copy of this consent document.

For adult participants:

Name and signature / thumb impression of the participant (or legal representative if

participant incompetent)

Name _________________________ Signature_________________

Date________________

Name and Signature of impartial witness (required for illiterate patients):

Name _________________________ Signature_________________

Date________________

Address and contact number of the impartial witness:

Name and Signature of the investigator or his representative obtaining consent:

Name _________________________ Signature_________________

Date________________