In partial fulfillment of the regulations for the award of the degree of M.D PATHOLOGY – BRANCH-III

DIAGNOSTIC VALUE OF GALECTIN - 3 IN THYROID NODULES A Dissertation submitted to

THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY, CHENNAI - 600032.

In partial fulfillment of the regulations for the award of the degree of M.D PATHOLOGY – BRANCH-III

DHANALAKSHMI SRINIVASAN MEDICAL COLLEGE AND HOSPITAL, SIRUVACHUR, PERAMBALUR - 621113.

May - 2020

ENDORSEMENT BY HEAD OF INSTITUTION

This is to certify that the dissertation entitled “DIAGNOSTIC VALUE OF GALECTIN – 3 IN THYROID NODULES” is a bonafide research work done by DR.DHIVYA.M under the guidance of DR.RATH.P.K, MD Professor of the Department of Pathology in partial fulfillment of the requirements of the degree of M.D Pathology and examination of The Tamilnadu Dr.M.G.R.

Medical University to be held in MAY –2020.

Date: Dean,

Place: Perambalur Dhanalakshmi Srinivasan Medical college and

Hospital,siruvachur

Perambalur - 621113

ENDORSEMENT BY THE HEAD OF THE DEPARTMENT

This is to certify that the dissertation entitled “DIAGNOSTIC VALUE OF GALECTIN- 3 IN THYROID NODULES” is a bonafide research work done by DR.DHIVYA.M under the guidance of DR.RATH.P.K, MD Professor of the Department of Pathology in partial fulfillment of the requirements of the degree of M.D Pathology and examination of The Tamilnadu Dr.M.G.R. Medical University to be held in MAY –2020

Date: Dr.UMADEVI T.B, M.D, Place: Perambalur Professor and Head,

Dept. of Pathology.

Dhanalakshmi srinivasan Medical college and Hospital,siruvachur

Perambalur - 621113

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “DIAGNOSTIC VALUE OF GALECTIN - 3 IN THYROID NODULES” is a bonafide research work done by DR.DHIVYA.M under my guidance in partial fulfillment of the requirements of the degree of M.D Pathology and examination of The Tamilnadu Dr.M.G.R. Medical University to be held in MAY –2020

Date Dr.RATH.P.K,M.D

Place: Perambalur Professor,

Dept. of Pathology Dhanalakshmi srinivasan

Medical college and Hospital,siruvachur

Perambalur - 621113

DECLARATION BY THE CANDIDATE

I, DR.M.DHIVYA hereby declare that this dissertation entitled

“DIAGNOSTIC VALUE OF GALECTIN - 3 IN THYROID NODULES” is a bonafide and genuine research work carried out by me under the guidance of DR.RATH.P.K, MD , Professor of the Department of Pathology, Dhanalakshmi Srinivasan Medical College and Hospital, Perambalur, Tamilnadu.

This dissertation is submitted to The Tamil Nadu Dr.M.G.R. Medical University, towards partial fulfillment of the requirement for the award of M.D Degree (Branch-III) in Pathology.

Date: Dr.Dhivya.M Place: Perambalur Postgraduate student

Dept. of Pathology,

Dhanalakshmi srinivasan

Medical college and

Hospital,siruvachur

Perambalur - 621113

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Date: Dr. Dhivya.M

Place: Perambalur Postgraduate student, Dept. of Pathology,

Dhanalakshmi srinivasan Medical college and

Hospital,siruvachur Perambalur - 621113

© The Tamilnadu Dr.M.G.R. Medical University, Chennai.

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This is to certify that this dissertation work titled “DIAGNOSTIC VALUE OF GALECTIN 3 IN THYROID NODULES” of the candidate Dr.Dhivya.M with registration Number 201713651 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 the introduction to conclusion pages and the result shows 17 percentage of plagiarism in the dissertation.

Date: DR. Rath.P.K,M.D, Place: Perambalur Professor,

Department of Pathology.

Dhanalakshmi srinivasan Medical college and Hospital,siruvachur

Perambalur - 621113

ACKNOWLEDGEMENT

I thank Professor Dr.Maragathamani elongovan M.S Dean, Dhanalakshmi srinivasan Medical College, for having permitted me to conduct the study and use the hospital resources in the study.

I express my heartfelt gratitude to Professor Dr. Umadevi.T.B MD, Professor and Head, Department of Pathology, Dhanalakshmi srinivasan Medical College, for her inspiration, advice and guidance in making this work complete.

I am greatly indebted to my guide Dr.P.K .Rath M.D ,Professor ,Department of pathology, Dhanalakshmi srinivasan Medical College , under whose inspiring and potential guidance,sustained interest and encouragement ,I was able to understand and finish this study

I would like to thank Dr.M.Suresh ,M.D ,Professor,Department of pathology

, Dhanalakshmi srinivasan Medical College for his encouragement

throughout my thesis

I am sincerely thankful to Dr.M.Elancheran ,M.D , Associate professor ,Department of pathology , Dhanalakshmi srinivasan Medical College for his valuable suggestions and contributions throughout my study

I wish to record my sincere gratitude to all Assistant professors ,Department of pathology Dhanalakshmi srinivasan Medical College for their constant support and encouragement during my study

I wish to thank all laboratory technicians, histopathology for their technical assistance.

I am grateful to my friends and my colleagues for the help they rendered during my study.

ABBREVIATIONS

CK - Cytokeratin

FA - Follicular Adenoma

FTC - Follicular Thyroid Carcinoma GAL 3 - Galectin 3

HBME-1 - Hector Battifora Mesothelial -1 IHC - ImmunoHistoChemistry

MIFC - Minimally Invasive Follicular Carcinoma MoAB - Monoclonal Antibody

PTC - Papillary thyroid carcinoma ROC - Receiver Operating Curve TPO - ThyroPeroxidase

TRH - Thyroid Releasing Hormone TSH - Thyroid Secreting Hormone TTF -1 - Thyroid Transcription Factor -1

WIFC - Widely Invasive Follicular Carcinoma

CONTENTS

CHAPTER

NO. TITLE PAGE NO

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 54

5 OBSERVATIONS AND RESULTS 64

6 ANNEXURE 1 – COLOR PLATES 79

7 DISCUSSION 84

8 SUMMARY 88

9 CONCLUSION 89

10 BIBILOGRAPHY 90

11. ANNEXURE II -MASTERCHART

1

.

INTRODUCTION

Thyroid nodules are more prevalant in the general population .Its occurance is related to the women sex, age, iodine deficiency and head and neck radiation. The rate of malignancy in thyroid nodules ranges from 1.5% to 17%. Although the risk of malignancy in any thyroid nodule is small, thyroid cancer carries the main differential diagnosis^1-6. Thyroid nodules, comprise benign and malignant neoplasms, as well as non neoplastic lesions such as colloid nodule and hyperplastic nodule⁷. The diagnosis of thyroid nodules can be established by histopathology and by clinical evidence in most of the cases. Some Thyroid lesions with nodular architecture and follicular pattern of growth often pose difficulties in accurate diagnosis^8. Hyperplastic nodules are recognized by differences in follicular size associated with degenerative changes. Follicular adenomas and normal parenchyma are separated by fibrous capsule.Presents as micro or macro follicular pattern along with lack of vascular lesions. In case of hyperplastic nodule presenting with mico or macro follicular pattern and adenoma with distortion of capsule there arises difficulty in diagnosis. Another difficulty is atypical follicular adenoma which have increased cellularity , nuclear atypia and mitotic activity. Another entity having difficulty in diagnosis is hyalinizing trabecular adenoma , which resembles the morphologic features of PTC⁸. Identification of benign lesion and accurate diagnosis is very important for proper management .Here arises the need for immunohistochemistry in differentiating between benign and malignant thyroid lesion.

1

Many authors have stressed the utility of immunohistochemistry in resolving the aforementioned diagnostic issues⁹.Galectin 3, betagalactosyl binding lectin may help in differentiating between benign and malignant thyoid nodules.

2

2. AIMS AND OBJECTIVES

1. To study the diagnostic value of Galectin 3 in thyroid nodules 2. To find the difference in expression of galectin 3 in benign and

malignant thyroid tumours

3

3.REVIEW OF LITERATURE ANATOMY

The thyroid gland is a bilobated organ connected by isthmus. The gland extends from fifth cervical vertebra to the first thoracic vertebra ¹⁰. The gland covered by capsule and pretracheal fascia ,which is responsible for the movement during deglutition.Left lobe of thyroid is smaller when compared to right lobe². The superior thyroid arteries, which branch from the external carotid arteries, and the inferior thyroid arteries, which branch from the subclavian arteries supply the thyroid gland¹⁰.

Embryogenesis of thyroid gland begins during fourth week of intrauterine life.Develops from foramen caecum as endodermal derivative and descent through the tongue carrying with it the thyroglossal duct. Thyroid gland secretes hormone from the twelth week¹⁰.

4

FIGURE - 1

ANATOMY OF THYROID GLAND

HISTOLOGY

Thyroid gland consists of thyroid follicles which is lined by single layer of cuboidal epithelium.Follicles contain homogeneous colloid.Thyroid gland surrounded by capsule from which septae arises dividing the gland into lobules¹¹.

5

FIGURE – 2

HISTOLOGY OF THYROID GLAND

PHSIOLOGY OF THYROID GLAND

The thyroid gland is a part of the hypothalamus-pituitary- axis(HPA).The hypothalamus secretes thyroid releasing hormone (TRH) which activates thyrotrophic cells of pituitary to secrete thyrotrophic secreting hormone (TSH) .Thyrotrophic secreting hormone acts on thyroid

follicular cells to produce thyroid hormones called triiodothyroxine (T3) and tetraiodothyronine or thyroxine (T4)¹².

The hormones (T3 and T4) control the secretion of both TSH and TRH by negative feed back mechanism¹².

6

IMMUNOHISTOCHEMISTRY OF THYROID

Thyroid transcription factor 1 (TTF1) expression is found in normal,benign and malignant thyroid follicular cells.Also expressed in lung epithelium.Shows diffuse positivity¹³

Thyroid transcription factor 2 (TTF 2) expressed in normal thyroid follicular cells 100% and also expressed in oropharynx,esophagus and tracheal epithelium¹³.

Paired box gene 8 is a member of the paired box (PAX) family .Expressed in thyroid tissue and kidney.Shows diffuse positivity with nuclear staining¹³.

Thyroglobulin which is the precursor molecule of thyroid hormones ⁸.It stains both cytoplasm and extracellular colloid¹³.Diffuse positivity seen in all normal thyroid follicular cells^13.

CK7, CK18, CK8 and CK19¹⁴.

Low molecular metal binding proteins such as ceruloplasmin, lactoferrin, transferrin, metallothionein¹⁵.

Vimentin¹⁴.

Follicular cells are joined by tight junctions containing the known components of these structures, including occludin and the various claudins¹⁶

Many pathologic lesions affect thyroid gland with diferent morphologies. Eventhough many lesions are there its to divide into two major types: one that shows a diffuse pattern and the other that produce nodules. Diffuse thyroid lesions are lesions in which entire thyroid gland is

affected, such as hyperplasias and thyroiditis. Nodular lesions are lesions which include both 7

non neoplastic hyperplasias as well as benign and malignant tumors¹⁷. Thyroid tumors are of two types; one that arises from thyroid follicular cells such as papillary carcinoma of thyroid , follicular carcinoma of thyroid and anaplastic carcinoma of thyroid and the Other one is Medullary carcinoma of thyroid that arises from thyroid parafollicular C cells.

Most of the tumors are are diagnosed by histopathological examination but sometimes it is difficult especially in distinguishing hyperplasia and follicular variant of papillary

carcinoma^.Differentiating follicular adenoma from follicular carcinoma are trouble some⁷. MULTINODULAR GOITRE

 Thyroid glands are enlarged irregularly due to repeated stimulation and involution

GROSS

 Simple goitre – cut surface shows firm in consistency and are amber

 Multinodular goiters are irregular and cutsuface filled with colloid and shows haemorrhage.Intact capsule are identified¹⁸

FINE NEEDLE ASPIRATION CYTOLOGY

 Cellularity of the smears varies from sparse to moderately cellular

 Sheets or macrofollicles and sometimes microfollicles are also seen

 Pigment laden macrophages along with thick or thin colloid also seen

 Nuclear features of papillary carcinoma such as overlapping ,pseudo inclusions and finely dispersed chromatin should be absent ^19,20

MICROSCOPY

 Follicles are dilated and in variable sizes lined with flattened epithelium 8

 Nodules are seen

 Fibrosis,calcifications,haemorrhage and osseous metaplasia – secondary changes that can be identified

 Papillary projections (Sanderson polsters) can be seen in few cystically dilated follicles

 It may mimick papillary carcinoma but nuclear features are absent ¹⁸

FIGURE – 3

MULTINODULAR GOITRE

Figure 3, Nodular with Variable sized follicles filled with colloid

9

FIGURE – 4

MULTI NODULAR GOITRE

Figure 4,Variable sized follicles lined by flattened epithelium HYPERPLASTIC NODULES

Non neoplastic hyperplasia usually presents with multiple nodules and can have dominant nodule,which become difficult to distinguish from true neoplasms GROSS

Thyroid enlarged with multiple nodules . secondary changes such as calcification,haemorrhage and cystic degenereation can be noted.

10

MICROSCOPY

Presents with different morphology .Some with many nodules lined by flattened epithelium and others with papillary projection which become difficult to distinguish from papillary

carcinoma¹⁸.

FIGURE – 5

HYPERPLASTIC NODULE

WHO classification of thyroid tumors - (2017)(21)

1. Tumors of the thyroid gland a. Follicular adenoma

11

b. Hyalinizing trabecular tumor

c. Other encapsulated follicular patterned thyroid tumors i. Tumors of uncertain malignant potential

ii. Noninvasive follicular thyroid neoplasm with papillary-like nuclear features d. Papillary thyroid carcinoma (PTC)

 Follicular variant of PTC

 Encapsulated variant of PTC

 Papillary microcarcinoma

 Columnar cell variant of PTC

 Oncocytic variant of PTC

e. Follicular thyroid carcinoma

 FTC, minimally invasive

 FTC, encapsulated angioinvasive

 FTC, widely invasive

i. Hürthle (oncocytic) cell tumors

 Hürthle cell adenoma

 Hürthle cell carcinoma

f. Poorly differentiated thyroid carcinoma 12

g. Anaplastic thyroid carcinoma h. Squamous cell carcinoma i. Medullary thyroid carcinoma

j. Mixed medullary and follicular thyroid carcinoma k. Mucoepidermoid carcinoma

l. Sclerosing mucoepidermoid carcinoma with eosinophilia m. Mucinous carcinoma

n. Ectopic thymoma

o. Spindle epithelial tumor with thymus-like differentiation p. Intrathyroid thymic carcinoma

q. Paraganglioma and mesenchymal / stromal tumors i. Paraganglioma

ii. Peripheral nerve sheath tumors

 Schwannoma

 Malignant PNST

iii. Benign vascular tumors

 Haemangioma

13

 Cavernous haemangioma

 Lymphangioma

iv. Angiosarcoma

v. Smooth muscle tumors

 Leiomyoma

 Leiomyosarcoma

vi. Solitary fibrous tumor r. Hematolymphoid tumors i. Langerhans cell histiocytosis ii. Rosai-Dorfman disease

iii. Follicular dendritic cell sarcoma iv. Primary thyroid lymphoma s. Germ cell tumors

 Benign teratoma

 Immature teratoma

 Malignant teratoma

t. Secondary tumors

14

Benign follicular thyroid tumors FOLLICULAR ADENOMA

Follicular adenoma is a common neoplasm of the thyroid gland and it is a benign encapsulated tumor. Clinically and biochemically patient will be euthyroid²². Follicuar adenoma lacks: (i) the evidence of capsular, vascular or any other type of invasion; and (ii) the nuclear features of the papillary family of neoplasms²³.

EPIDEMIOLOGY

In multi nodular goitre usually pathologist donot make a diagnosis of follicular adenoma ,usually designed as hyperplastic nodules when there is no eviodence of malignancy.Follicular adenoma has a clonal origin while hyperplastic nodule is polyclonal origin⁷⁴.

Incidence of follicular adenoma is 3 -5%.Incidence is more commin in iodine deficiency areas.Most commonly affects female and all age group is affected (mostly fifth and sixth decade)⁷⁴.

CLINICAL FEATURES

Patient with large tumor develops symptoms of dyspnea, coughing , hoarseness, or dysphagia due to compression of trachea,recurrent laryngeal nerve and esophagus. Sometime present with hyperthyroidism²²

15

ETIOLOGY

Iodine deficiency and endemic goiter are the predisposing factors associated with follicular adenoma³¹.Radiation exposure increases the risk of follicular adenoma by 15 times⁷⁴.The

PAX8-PPAR gene rearrangement has been found in 4-13% of follicular adenomas.RAS mutation has also been found which plays an important role in transferring adenomas to follicular

carcinoma^22,24.

FINE NEEDLE ASPIRATION CYTOLOGY

Characterised by many follicular epithelial cells in sheets and in microfollicle pattern,3 dimensional clusters and in isolated in a background of scanty or no colloid^22,25. Hyperplastic nodule can be differentiated from follicular neoplasm by presence of cohesive follicular cells ,macrofollicles and basement like material while follicular neoplasm has loosely cohesive clusters and clean background with absence of basement membrane or matrix like material^26.27.

MACROSCOPY

A follicular adenoma is encapsulated tumor with solitary round or oval nodule, ranges from 1-3 cm. ²¹. The capsule can be thin or thick. The cut surface is homogeneous greyish-white, tan and brown in colour. Secondary changes such as haemorrhage and cystic degeneration can also be noted.Multiple tumrs are usually rare⁷⁴.

MICROSCOPY

 Completely enveloped by thin fibrous capsule . 16

 Capsular and vascular invasion is absent.

 They are classified based on their growth pattern as macrofollicular, simple, microfollicular, fetal, embryonic and trabecular³⁴

 The tumour cells can be cuboidal , polygonal or tall . The cytoplasm is

moderately abundant with pale eosinophilic to amphophilic. The nuclei is round with basally located and even chromatin distribution⁷⁴

 Lacking the nuclear features of papillary thyroid carcinoma²⁰

FIGURE -6

FOLLICULAR ADENOMA

Figure 6 ,Tumour cells are completely enveloped by fibrous capsule

17

IMMUNOPHENOTYPE

Follicular adenomas are immunoreactive for

 cytokeratins,

 thyroglobulin

 TTF1

 PAX8

 Negative for calcitonin and carcino embryonic antigen

 Immunoreactivity for galectin 3 and HBME is very rare

FIGURE – 7

FOLLICULAR ADENOMA

Figure 7,The tumour cells are cuboidal and the cytoplasm is moderately abundant with pale eosinophilic and the nucleus is round

18

VARIANTS

 Follicular adenoma with papillary hyperplasia

 Follicular adenoma with bizarre nuclei

 Lipoadenoma

 Signet-ring cell follicular adenoma

 Clear cell follicular adenoma

 Spindle cell follicular adenoma

DIFFERENTIAL DIAGNOSIS

 Hyperplastic/adenomatoid nodules¹⁷

 Follicular carcinoma³⁰

 Follicular variant of papillary carcinoma

 Atypical follicular adenoma¹⁷

PROGNOSIS AND PREDICTIVE FACTORS

Complete removal of follicular adenoma carries no risk⁷⁴.

‗‗

ATYPICAL FOLLICULAR ADENOMA’’

 Encapsulated follicular lesions,

 Lack evidence of capsular or vascular invasion

 Shows cellularity,necrosis,haemorrhage and infarction³¹ 19

HYALINIZING TRABECULAR ADENOMA Epidemiology

Most commonly occurs in female with mean age of 50 years

Etiology

Radiation exposure has been reported occasionally CLINICAL FEATURES

Usually asymptomatic

MACROSCOPY

Single,

well-circumscribed or encapsulated tumour with round or oval shape. Cut surface shows white in colour and soft to firm in consistency.

MICROSCOPY

 Well circumscribed

 Trabecular or zell ballen or lobulated

 Cells are large or medium size and polygonal

 Cytoplasm – mostly eosinophilic.granular and rarely perinuclear halo

 Nucleus – round,vesicular,grooves and membrane irregularities are also seen

 Mitosis are rare

 Absence of capsular ,vascular and parenchymal invasion 20

 Abundant hyaline amorphous material is present within the trabeculae (congo red is negative)

MALIGNANT NEOPLASM 1.PAPILLARY CARCINOMA

Papillary carcinoma accounts for 80–85% of malignancy of thyroid²⁸.Have excellent prognosis and 10% recurrences occur²⁹.Occurs more commonly in women

EPIDEMIOLOGY

 Common cancer in both adults and children.



Accounts for about 65% of cases in Ireland, 86.2% in the USA, and 93% in Japan



The median patient age at diagnosis is 50 years

 Women incidence is three times more than men.⁷⁴

ETIOLOGY

Etiological factors are

 Environmental

 Genetic

 Hormonal factors

 Radiation^28,34

LOCALIZATION

 Can arise in either lobe or in the isthmus 21

 Can also occur in ectopic thyroid⁷⁴

GENETIC FEATURES

18,24,32,33

 BRAFp.V600E

Most common alteration seen and very good diagnostic factors for diagnosis of papillary thyroid carcinoma

 PAX8/PPARG

Noted in 20-50% of papillary carcinoma

 RET/PTC

Fusion oncogene .Most common will be gene rearrangement.It is a transmembrane tyrosine kinase

 TERT

Represents poor prognosis

 ALK

Rearrangement noted in 1-5% of papillary carcinoma

 RAS

Most commonly seen in follicular variant of papillary carcinoma

CLINICAL FEATURES

 Usually presents as an asymptomatic mass with or without enlargement of regional (cervical) lymph nodes

 In approximately 20% of cases Hoarseness and dysphagia occur 22

 Nodal metastases in the lateral neck are reported in 27% of patients at presentation⁷⁴

FINE NEEDLE ASPIRATION CYTOLOGY

 Papillae, sheets, microfollicles

 Nuclear changes

o Nuclear enlargement

o 'Powdery' chromatin³⁵

o Nuclear pseudoinclusions and grooves

o Nucleoli (small or large)³⁶

o Irregular nuclear membrane

 Nuclear crowding and molding

 Variable cytoplasm (scant, squamoid, hürthle-like, vacuolated)³⁷

 Psammoma bodies

 Histiocytes (multinucleated giant cells)

MACROSCOPY



Grey white



firm in consistency



calcification and cystic change also noted²⁸

23

MICROSCOPY



many papillae which are complex ,branching and randomly oriented with central fibro vascular core



papillae lined by single or many layer of cuboidal cells



Extensive fibrosis is noted



Psammoma bodies – concentric lamellation with basophilic in appearance^18,28,38

NUCLEAR FEATURES

 optically clear nucleus – large size and overlapping of nucleus seen.The nucleolus is inconspicuous .Also called as orphan annie eyed or ground glass nucleus

 Nuclear pseudoinclusion – invagination of cytoplasm in the nucleus.

 Nuclear grooves – due to infoldings of nuclear membrane .seen in the longest nuclear axis

 Some of them may have solid or trabecular pattern spindle cell component also identified^18,28

24

FIGURE -8

PAPILLARY CARCINOMA

Figure 8, Papillae with fibrovascular core

HISTOLOGICAL TYPES

1.PAPILLARY MICROCARCINOMA

 papillary carcinoma with 1cm or less in diameter .Usually not identified by FNAC due to its small size.Have good prognosis and rare metastasis¹⁸

 Also called occult sclerosing carcinoma and occult papillary³⁹

 Lesions are mostly missed in gross bcoz of its small size^40,41

 Microscopically, has scar like appearances 25

 The neoplastic elements predominate at the periphery, whereas other elements are entrapped in the centre.

 Covered by fibrous capsule

 Overall, the prognosis of papillary microcarcinoma 1s excellent.

 Prognosis is good³⁰

2.ENCAPSULATED VARIANT

 Tumour entirely Surrounded by capsule⁴² .

 Differ from normal papillary carcinoma by having normal or hyperchromatic nucleus .Have potential to metastasis

 Accounts for 10% of cases

 Prognosis is good⁷⁴

3.FOLLICULAR VARIANT

Papillary carcinoma containing more or less completely follicles with similar nuclear features of papillary carcinoma⁴³.

26

FIGURE – 9

FOLLICULAR VARIANT OF PAPILLARY CARCINOMA

Figure 9,enlarged and overlapped vesicular nuclei with irregular membranes ,bright colloid with scalloping(H&E ,high power)

Infiltrative variant – with infiltrative margins

Encapsulated follicular variant – surrounded by capsule completely

Noninvasive follicular thyroid neoplasm with papillary like nuclear features (NIFTP) –

 Full encapsulation or partial encapsulation with clear demarcation

 Growth pattern (<1% papillae)

 Nuclear features of papillary carcinoma, defined by a nuclear score of 2 or 3

 Absence of lymphovascular and capsular invasion⁴⁴

 Less than 30% of the tumor volume showing a trabecular, insular or solid architecture 27

 Less than 3 mitoses/10 high power fields

 No necrosis identified

 No psammoma bodies.

4.SOLID VARIANT

 Most common in children and young adults

 Proliferation of follicular cell occurs more than secretory activity seen as solid nests.

 1-3% of papillary carcinoma belongs to solid variant

 Metastasis to lung more common

 Poorly differentiated carcinoma resembles solid variants but it does not exhibit nuclear features of papillary thyroid carcinoma and has high mitotic activity and necrosis

 Aggressive behaviour³⁴

5.MACROFOLLICULAR VARIANT

Proliferating activity is less than secretory activity so follicles appears dilated²¹

6.DIFFUSE SCLEROSING VARIANT

 Most common in women,

 Most frequently in the second or third decade of life

 Diffuse enlargement of the thyroid gland is seen 28

 Dense sclerosis associated with diffuse involvement of the gland.

 Psammoma bodies are noted

 Tumour nests appear solid, with associated squamous metaplasia.

 Metastasis are common^28,38

7.ONCOCYTIC VARIANT

 Very rare tumour

 Have abundant and granular cytoplasm

 Prognosis is good

8.TALL CELL VARIANT

 Contains papillae lined by cells having height three times that of width

 Tram track or cord pattern also identified in addition to papillae

 Tumor shows abundant eosinophilic (oncocytic-like) cytoplasm.

 Nuclear features and pseudo inclusions are also noted

 Tall cells accounting for 30% of tumor cells are required for the diagnosis of the tall cell variant

 The tall cell variant usually occurs at an older patient age

 Aggressive variant

 Prognosis is less favourable^30,43,44

29

FIGURE – 10

TALL CELL VARIANT OF PAPILLARY CARCINOMA

Figure 10,Tram track pattern(H&E,X40)

9.COLUMNAR CELL VARIANT

 Rare variant

 Lined by columnar cells showing pseudostratifications

 Cells lack the conventional nuclear features of Papillary carcinoma

 Prognosis is poor .^28,38

30

FIGURE – 11

COLUMAR CELL VARIANT OF PTC

Figure 11,stratified tall eosinophilic epithelium with elongated nuclei,psommoma body noted (H&E,high power)

10.HOBNAIL VARIANT

 >30 of cells show hobnail features

 Rare variant

 Papillary arrangement with follicular cells containing abundant eosinophilic cytoplasm and apically placed nuclei with prominent nucleoli

 Psammoma bodies are present 31

 Necrosis, mitoses , angiolymphatic invasion, and extrathyroidal extension are common.

 Recurrence and metastasis to lymph nodes are common

 Have aggressive behaviour28,38,43,44

11.CRIBRIFORM-MORULAR VARIANT

 Cribriform and morular pattern of growth.mostly seen in females

 Sporadic cases are usually solitary and cases associated with familial adenomatous polyposis are multifocal.

 Encapsulated tumor

 cribriform, follicular, papillary, trabecular, and solid growth patterns, with round squamoid structures called morules.



The morules contain optically clear nuclei ²⁸

SPREAD AND METASTASIS

 Lymphnode metastasis are common than blood bourne metastasis

 cervical lympphnode are common and extrathyroidal soft tissues are also affected

 metastsis to lung common through blood bourne.can also occurs in breast , bone, soft tissues and CNS.^30,43

PROGNOSIS

 Male have worst prognosis than female 32

 Size of the tuour inverse relationship with prognosis

 Multicentricity prognosis poor

 Lung metastasis have poor prognosis

 Extrathyroidal extension poor prognosis

 Lymphnode metastasis poor prognosis

 Squamous,anaplastic and poorly differentiated areas if present will be of poor prognosis

 TERT and BRAFv600E have poor prognosis18,28,38,43,44

FOLLICULAR THYROID CARCINOMA (FTC)

Carcinoma with evidence of vascular or capsular invasion and absence of nuclear features of papillary thyroid carcinoma^22,31

Second most common malignancy of thyroid 5-10%^22,31

Follicular carcinoma cannot be differentiated from follicular adenoma by cytology

ETIOLOGY

²²

Iodine deficiency and endemic goitre are known predisposing factor for follicular carcinoma.

GENETIC FEATURES

RAS gene occur in 30%to 50% of follicular carcinomas

PAX8-PPARγ gene fusion - results in the production of fusion protein (PPFP) . 33

PPFP is found in follicular carcinoma . TERT – 10 – 35% of follicular carcinoma

PIK3CA and PTEN – in 0 – 10% of follicular carcinoma^18,24

FINE NEEDLE ASPIRATION CYTOLOGY

 Moderate to high cellularity^26.27

 Repetative microfollicles

 Cells are equal sized arranged in syncytium or rosettes^19,20

 Overlapping and crowding of nucleus seen

 Scanty or no colloid

MACROSCOPY

Follicular carcinoma have thick capsule with round to oval in shape^18,45.

Macroscopically, the tumors are light brown to pink in cut section, with areas of haemorrhage, necrosis and fibrosis. Kuru et al ⁴⁵ found that nodule size more than or equal to 4cm was associated with increased risk of malignancy compared with nodule size less than 4cm.

MICROSCOPY

 Varies from well formed follicles to poorly formed follicles or solid growth pattern

 Even cribriform areas and trabecular formations or spindle cells are noted.

34

 Microscopically, these carcinomas are quite uniform and hyper cellular, showing follicle formation with minimal colloid, while a widely invasive subtype often shows a solid or trabecular growth pattern.

 The tumor cells lack the nuclear features of papillary carcinoma of thyroid.

 The hurtle cell variant of follicular thyroid carcinoma is composed of oxyphilic cells with a typical appearance of abundant granular and eosinophilic cytoplasm^46-53 .

 The presence of vascular and/or capsular invasion is the only feature that can distinguishes a follicular carcinoma from follicular adenoma, which means that distinction between the two requires thorough inspection of the tumor capsule interface⁴⁶ Follicular carcinoma invasion ;

Histological confirmation of capsular invasion and /or vascular invasion is necessary for the diagnosis of Follicular Thyroid Carcinoma.

Capsular invasion

 Complete transgression of the fibrous capsule (the tumor has to penetrate the entire thickness of the capsule) should be considered as an unequivocal evidence of capsular invasion

 But the invading tumor nests should show a connection with the main tumor mass^31,46-53

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FIGURE - 12

FOLLICULAR NEOPLASM WITH FIBROUS CAPSULE

The A. Inner aspect of the capsule showing bosselation is not a capsular invasion

B. Sharp tumor bud invades into but not through the capsule suggesting invasion requiring deeper sections to exclude.

C. Transgression of capsule beyond the outer contour of the capsule considered as capsular invasion

D.Tumor that have developed a thin fibrous capsule but already extending beyond an imaginary (dotted) line drawn through the outer contour of the capsule considered as capsular invasion

E. Satellite tumor nodule features same as tumour cells presents outside capsule is considered as capsular invasion

36

F. Follicles arranged perpendicular to tumour does not represent capsular invasion G. Follicles arranged parallel to the capsule do not represent capsular invasion H. Mushroom-shaped tumor with total transgression of the capsule represents capsular invasion

Vascular invasion

 Tumour embolus should be present in the capsular vessels which is adherent to the vessel wall and the endothelial cells

The histological criteria for diagnosis of vascular invasion are defined as follows:

1.In subendothelial location tumour should be present as plug or polyp

2. Endothelium should cover the tumour completely.tumour cells lying in the lumen will be a artefact

3. The endothelialised tumor embolus should be attached to the vessel wall ³¹ Vascular invasion is the more consistent feature of malignancy than

capsular invasion

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FIGURE – 13

VASCULAR INVASION

A.Tumor fragments free floating inside the vessel usually represents artifacts not considered as vascular invasion

B. Tumor bulging and indenting the vessel wall are not counted as vascular invasion C.Tumor floating in intracapsular vessel may be from sectioning of tumor is not counted as vascular invasion

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D. Endothelialized tumor deposit is juxtaposed to the vessel wall not couned as vascular invasion.

E. Tumor is juxtaposed to vessel wall and associated with a thrombus.

F. Tumor penetrating vessel wall also with thrombus formation

G. Tumor fragments in intermingled with an organized thrombus and adherent to vessel wall.

SUBTYPES

MINIMALLY INVASIVE (LOW-GRADE) FOLLICULAR CARCINOMA

which in turn can be Subdivided into: -

1. Have only capsular invasion, without angioinvasion 2. With angioinvasion

 limited angioinvasion, including less than 4 vascular spaces

 Extensive angioinvasion,- including 4 or more than 4 vascular spaces.51

WIDELY INVASIVE FOLLICULAR CARCINOMA

 shows widespread infiltration of the tumor into the thyroid parenchyma and into the blood vessels

 often lack complete encapsulation

 Penetration of entire thickness of the capsule by the tumor with or without vascular permeation.^51,52,53

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Follicular Carcinoma is further classified based on the tissue pattern as follows;

PROGNOSIS

 Follicular carcinomas that contains capsular invasion without vascular invasion carries a good prognosis

 Number of vessels involved directly proportional to the,more number of vessels worst prognosis

 Most common metastasis sites are bone ,lung brain and liver

 TERT mutation is an independent marker of a higher risk of disease reccurances⁵⁴

FOLLICULAR CARCINOMA ONCOCYTIC TYPE (HURTHLE CELL VARIANT)

EPIDEMIOLOGY

 More common in men

 Most common occurs in older age ( 57 years)

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CLINICAL FEATURES

 Usually painless mass

 Rapid enlargement of the tumour resulting in respiratory compromise.

MACROSCOPY

 Grossly brown to mahogany.

 Capsule is usually seen

MICROSCOPY

Large cells with voluminous granular cytoplasm along with centrally located nuclei, which often have prominent nucleoli⁷⁴

Nests and clusters of tumorcells are seen with fibrous bands Solid, trabecular, and follicular pattern of growth is identified

PROGNOSIS AND PREDICTIVE FACTORS

Prognosis depends on vascular invasion ,the more veins invaded by tumour, the less favourable the prognosis. Larger size (≥4cm strongly correlated to malignancy Higher prevalence of aggressive behavior and Lower survival rate ⁵⁴

MEDULLARY CARCINOMA

 Arises from para follicular cells which secretes calcitonin.It is a tumour of neuroendocrine origin

 Usually rare tumor 1 - 2% of thyroid carcinomas

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 Either sporadic (nonhereditary) or familial (hereditary)

Sporadic:

 constitutes 70% of medullary carcinoma

 Usual age will be 45 years

 Solitary always

 Mutation of RET oncogene in 40-50%

 RAS mutation are also seen Familial:

 Constitute 30% of medullary carcinoma

 Usually age will be around 35

 Often multiple and bilateral

 Can occur in one of the three 1. MEN 2A

2. MEN 2B syndromes,

3. familial medullary thyroid carcinoma (FMTC) syndrome

 Caused by gain of function germline mutations in the RET ^18,55

FINE NEEDLE ASPIRATION CYTOLOGY

 Cellular smears

 Clusters or singly scattered

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 Can be spindle or plasmacytoid

 Binucleate and multinucleated cells

 Stippled nuclear chromatin

 Amyloid – amorphous may be pink or violet

 Positive staining for calcitonin^19,20

MACROSCOPY

 Usually solid and firm

 Can be well circumscribed or non encapsulated

 Cut surface revealed grey to yellow

 Fibrous capsule enveloping the whole tumour can also be identified^18,55

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FIGURE – 14

MEDULLARY CARCINOMA THYROID

Figure 14,medullary carcinoma thyroid with background of amyloid

MICROSCOPY



Solid sheets of polygonal to round cells with abundant amphophilic and granular cytoplasm and medium sized nucleus



Amyloid and collagen seen in the background^61,63



Growth can be trabecular ,glandular, pseudopapillary,carcinoid like or paraganglioma like



Psammoma bodies are sometimes seen



Tumour cells exhibits various shape ,may be plasmacytoid,spindle ,squamoid or squamous

44



Can be

1. Oncocytic – amphophilic rather eosinophilic 2. Papillary – papillary form

3. Mucinous 4. Clear cell variant

5. Small cell – resembling small cell lung carcinoma 6. Pigmented variant^58,59,60

IMMUNOHISTOCHEMISTRY

 Positive for chromogranin A and B

 Synaptophysin

 TTF1

 Keratin

 Calcitonin

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FIGURE – 15

CHROMOGRANIN POSITIVE

Figure 15,chromogranin positive in medullary carcinoma thyroid

POOR PROGNOSTIC FACTORS

 older age

 advanced stage

 the presence of lymph node metastasis at diagnosis



somatic RET mutation¹⁰

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SPREAD AND METASTASIS

 Can invade locally

 Can metastases to cervical and mediastinal lymphnode

 Distant organ such as lung ,liver and skeletal system are involved18,27,55-64

ANAPLASTIC CARCINOMA

 Extremely aggressive and undifferentiated tumour

 Usually occurs in elderly with rapidly growing mass

 Presents with difficulty in breathing and swallowing and hoarsness of voice GENETIC

 TP53

 TERT

 RAS

 BRAFp.V600E

 PIK3CA

 PTEN

TP53 will be mostly associated with anaplastic carcinoma .

FINE NEEDLE ASPIRATION CYTOLOGY

 Multinucleate or bizaree cells

 Can be spindled or squamoid

 Atypia and mitosis almost seen

 Background shows necrosis^46,47

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MACROSCOPY

Mostly solid tumour with areas of necrosis and haemorrhage are noted.No other specific features particular to this tumour

MICROSCOPY

 It doesnot makes any pattern but epithelial in appearance and in immunohistochemistry

 Includes squamoid and sarcomatoid

 Squamoid – foci of keratin

 Sarcomatoid – spindle cells and giant cells

 Fascicular or storiform pattern

 Osteoclast like multinucleated giant cells are seen

FIGURE 16

ANAPLASTIC CARCINOMA THYROID

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VARIANT

 Rhabdoid variant

FIGURE – 17

ANAPLASTIC CARCINOMA – RHABDOID VARIANT



Paucicellular variant – fibrosis and hyalinization noted

IMMUNOHISTOCHEMISTRY



Keratin – positive



Vimentin – positive



Thyroglobulin – Negative



PAX 8 – positive



TTF1 and TTF2 – focally positive

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PROGNOSIS

Median survival is 6 months ²⁷

TNM CLASSIFICATION (AJCC 8

^TH

EDITION)

Primary Tumor (pT)

pTX: Primary tumor cannot be assessed pT0: No evidence of primary tumor

pT1: Tumor less than 0r equal to two cm in greatest dimension, but limited to the thyroid pT1a: Tumor less than or equal to one cm in greatest dimension,but limited to the thyroid pT1b: Tumor more than one cm but less than or equal to two cm in greatest dimension,but limited to the thyroid

pT2: Tumor more than two cm, but less than or equal to four cm in greatest dimension, but limited to thyroid

pT3: Tumor more than four cm limited to the thyroid, or gross extrathyroidal extension invading only strap muscles

pT3a: Tumor >4 cm limited to the thyroid

pT3b: Gross extrathyroidal extension invading only strap muscles (sternohyoid, sternothyroid, thyrohyoid, or omohyoid muscles) from a tumor of any size

pT4: Includes gross extrathyroidal extension beyond the strap muscles

pT4a: Gross extrathyroidal extension invading subcutaneous soft tissues, larynx, trachea, esophagus, or recurrent laryngeal nerve from a tumor of any size

pT4b: Gross extrathyroidal extension invading prevertebral fascia or encasing the carotid artery or mediastinal vessels from a tumor of any size

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GALECTIN 3

Galectin-3 is a beta-galactoside binding polypeptide belongs to lectin family.Molecular weight will be around 30kDa ^8,9,13

FUNCTION

^8,9

Play a significant role in a number of biological processes.

 Cell-cell and cell-matrix interaction

 Linkage of cell

 Movement of cell

 Damaged cell repair.

 Inflammation

 Neoplastic transformation.

EXPRESSION

 Human macrophages and neutrophils, mast cells, and Langerhans cells shows Galectin-3 expression.



colorectal and breast cancer shows downregulated expression⁶⁶



Shows cytoplasmic positivity and nuclear positivity8,9,13,65,66,67

REGULATION OF APOPTOSIS

In normal state Galectin 3 will be upregulated during cell growth .While in disease ,papillary carcinoma Galectin3 will be over expressed

It causes increased expression of nuclear transcription factor and to high proliferative state of the cell

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Reduced expression of galectin 3 causes apoptosis in papillary carcinoma of thyroid^68-72. p53-mediated apoptosis is regulated by Galectin 3 expression. Positive correlation between p53 mutations and Gal-3 expression in human thyroid carcinoma cells were identified.

Cellular Transformation and Metastasis

Studies revealed that overexpression of Galectin-3 in colon cancer causes liver metastasis while downregulation disfavours metastasis. It also suggest that Galectin-3 helps in regulation of normal cell proliferation and overexpression of Galectin-3 results in malignant transformation and metastasis.^67,83,84

Distribution and intensity of Gal-3 cytoplasmic signal interpretation was based on the guidelines followed by Weber KB et al⁸⁵., and Hermann ME⁷³ et al.,

Intensity of staining was graded as 0 to 3 0 – No staining

1+ - weak staining 2+ - Moderate staining 3+ -Intense staining

Stained cell proportion interpreted as 1+ - (< 5% of cells)

2+ - (5% to 50% of cells)

3+ - (>50% of cells) 52

Many studies reported Gal -3 positivity in 90% to 100% of papillary carcinoma cases . Few studies reported Gal-3 expression in PTC varies by histological subtype, 82% to 100% cases of classic variant of papillary carcinoma shows positivity . 75% Gal-3 positivity expression is noted in FVPTC. No significant difference was found between FVPTC and classic PTC for the expression of Gal-3 in the study of 181 PTC‘s by Cvejic et al., Gal -3 expression was from 20% to 100% in cases of FTC . Few study, Gal -3 expression found positive in 50%

cases of MTC .Studies have noted variable Gal-3 expression in poorly differentiated thyroid cancers also .^46,67

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4. MATERIALS AND METHODS

This prospective and retrospective study material includes the patient who underwent surgery at Dhanalakshmi srinivasan medical college for nodular thyroid disease with histopathological diagnosis of non neoplastic and neoplastic thyroid lesions using haematoxylin and eosin staining in the Department of Pathology, Dhanalakshmi srinivasan Medical College during the period of June 2014 to June 2019

4.1) Study design Prospective study

4.2) Inclusion criteria

All Nodular thyroid lesion with histopathological confirmation

4.3) Exclusion criteria

a) Non nodular thyroid lesions diagnosed on histopathological evaluation b) Block could not be retrieved

c)Insufficient tissue in the block for performing IHC.

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4.4 ) Sample size

A total number of 50 cases of surgically resected thyoid lesions that includes non neoplastic lesions, benign and malignant thyroid tumors were collected.

Out of 50 cases 28 were non neoplastic lesions that includes colloid nodule,multinodular goitre and adenamatoid nodule ,8 were benign that includes follicular adenoma (n = 8) and 14 were malignant that includes papillary carcinoma of all variants (n = 12) and follicular carcinoma (n = 2).

4.5)Materials required

(1) Formalin fixed paraffin blocks are obtained from all the cases of nodular lesions of the thyroid

(2) Hematoxylin and eosin stained tissue sections with the blocks of nodular thyroid lesions (3) Slides of positive charge are required for IHC

(4) chemicals which are used for preparing antigen retrieval and was buffer (5) For antigen retrieval pressure cooker is also needed

(6) Primary antibodies ( GALECTIN 3) and universal kit are needed (7) Microscope for grading in IHC

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4.6 METHODOLOGY

COLLECTION OF BLOCKS AND SLIDES

Patient age, sex and type of thyoid surgery were collected from the case sheets of the patient and from the medical records of dhanalakshmi srinivasan medical college

The diagnostic criteria taken are as follows

1.Colloid nodule – varying sized follicles lined by flattened epithelium filled with colloid Nuclear features of papiilary thyroid carcinoma should be absent

2.Adenamatoid nodule – Diagnosed by usually unencapsulated mass along with presence of secondary changes.

3.Multinodular goitre – Multiple nodules having varying sized follicles lined by flattened epithelium to cuboidal epithelium .Nuclear features of papillary carcinoma should be absent.Fibrosis,calcification and haemorrhage are the secondary changes that can be identified

4.Follicular adenoma – Diagnosed by encapsulated mass along with proliferation of follicles and there should be absence of nuclear features of papillary carcinoma and absence of vascular/capsular invasion

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5.Follicular carcinoma - Diagnosed by capsular/ vascular invasion along with atypical nuclei and that lacks the nuclearvfeatures of papillary carcinoma

6.Papillary carcinoma,classic variant – diagnosed by presence of branching papillae with fibrovascular core .Nuclear features such as clearing of the nucleus ,nuclear grooves and pseudo nuclear inclusions can be identified.psommamo bodies can also be identified

7. Papillary carcinoma ,follicular variant - Papillary carcinoma containing more or less completely follicles with similar nuclear features of papillary carcinoma

CONTROL BLOCKS

Tissues of papillary thyroid carcinoma,classic variant is taken as positive control for Galectin - 3

PREPARATION OF HAEMATOXYLIN AND EOSIN SLIDES

 Blocks of all cases of thyroid nodules are collected

 Tissues were fixed in the formalin to maintain the structure of the tissues

 Then dehydration in ascending series of alcohol is done

 Followed by clearing by xylene

 Impregenation with paraffinwax

57

 Embedding is done , a process by which tissues are provided with a supporting medium for cutting .Done with paraffin wax

 Tissues are cut with a microtone in 3- 4 microns

 Paraffin is dissolved with a solvent

 Rehydration done with descending alcohol series

 Slides were stained in haematoxylin

 Blueing was done

 Counterstained with1% eosin

 Mounted in DPX

 Haematoxylin stains the nucleus blue

 Eosin stains the cytoplasm pink

PREPARATION OF IHC SLIDES

Slide cutting and antigen retrievel

 Sections were taken at thickness of 4-5 microns on the surface of the APES (3- aminopropyltriethoxysilane) coated slides.

 Incubation was done for 1 hour at 60-70degree.

 Antigen retrievel solution and wash buffer was used as prescribed by the manufacturer (PATH INSITU)

 The buffers are

1. Tris EDTA buffer at a PH of 9 2. Tris wash buffer at PH of 7.6.

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BUFFER PREPARATION TRIS EDTA BUFFER

Tris –6.05gm EDTA--O.744gm 1 N HCL—4ml Distilled water—1litre TRIS WASH BUFFER

Tris -6.05gm

Sodium chloride -8gm 1N HCL-4ml

Distilled water-1 litre PRECAUTIONS

a) The glassware of immunohistochemistry must be clean and dry

b) The buffer used should be prepared fresh and adequate pH must be achieved by adjusting accordingly.

59

c) During the process of immunohistochemistry , the slide should never be

allowed to dry, and so a humidity chamber is used during staining and incubation.

d) DAB chromogen should be handled carefully as it is potentially carcinogenic.

e) All reagents –primary antibody ,DAB chromogen, Peroxidase block should be stored at ideal 4- 6 degree celcius

f) Every batch of slide should accompanied by positive and negative controls which ensures quality of the procedure.

PROCEDURE Antigen retrieval;

Many methods have been used for antigen retrieval which includes Microwave method, and water bath, autoclave, proteolytic enzyme and pressure cooker method. In our institution we followed antigen retrieval by using pressure cooker method ,as it produces even heating with lesser disadvantages as compared to other methods.

60

Procedure for immunohistochemistry as given by manufacturer;

1. Section cutting and incubation is followed by Xylene wash (3 changes) for 15minutes each.

2. Rehydrated in graded alchohol containing 100%, 80%, 70% for five minutes each.

3. Rinsed in distilled water for 2minutes each.

4. Antigen retrieval for 15-20 minutes in Tris-EDTA buffer and ph of the retrievl buffer adjusted to 9.

5. washed in distilled water, two changes, 2 minutes each.

6. Washed in TRIS wash buffer- 3changes 5minutes each.

7. Do endogeneous peroxidase blocking by adding H2O2 on the section, for 7 - 10minutes 8. Washed in TRIS wash buffer- 3changes 10minutes each.

9. Application of primary antibody ( GALECTIN 3) – 30 mins in moist chamber.

10. Washed in TRIS wash buffer- 3changes 10minutes each.

11. Add polyexcel Target binder for 15 mins

12. Washed in TRIS wash buffer- 3changes 10minutes each 61

13. Application of HRP POLYMERASE for 15 mins.

14. Washed in TBS wash buffer- 3changes 10minutes each.

15. Application of Diamino-benzidine tetrachloride(DAB) chromogen (1 drop) and DAB buffer (1ml) for 5 mins.

16. Washed in distilled water – 2 changes.

17. Counterstaining with Harris Hematoxylin – 1dip/30seconds to impart background staining.

18. Wash in running tap water.

19. Dehydrate (70%,80%,100%) 5minutes

20. Clear with xylene – 2 changes 5 minutes each.

21. Mount the section with Dextrene Phthalate Xylene 22. Observation and grading under light microscope.

ANTIBODIES USED

Antibodies Clone Dilution Manufacturer Buffer for antigen retrievel

PH of TRIS buffer

Visualisation kit

manufacturer Galectin 3 b2c10 Pre

diluted

Pathin situ TRIS EDTA

9 Pathin situ

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4.7 GRADING OF IHC(Galectin 3) STAINING

 Cytoplasmatic ± nuclear immunoreactivity for Gal-3,

 Irrespective of intensity of staining,more than 5% of cells stained is taken as positive

Intensity of staining of cells;

1 = weak 2 =moderate 3 = strong

Proportion of cells stained

1+ (< 5% of cells)

2+ (5% to 50% of cells) 3+ (>50% of cells)

63