Swetha p j

I

“COMPARATIVE STUDY OF MODIFIED ULTRAFAST PAPANICOLA OU

STAIN AND RAPID ECONOMIC ACETIC ACID PAPANICOLAOU S TAIN

WITH THE ROUTINE STAINS USED IN CYTOLOGY”

Submitted by

DR. P.J.SWETHA

Dissertation submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

In partial fulfillment of the requirements for the degree of

DOCTOR OF MEDICINE

In

PATHOLOGY

Under the guidance of

DR.KUSUMA VENKATESH MD, DCP. PROFESSOR

DEPARTMENT OF PATHOLOGY KEMPEGOWDA INSTITUTE OF MEDICAL SC IENCES, BANGALORE- KARNATAKA 2013

II

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA.

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation/thesis entitled

“COMPARATIVE STUDY OF MODIFIED ULTRAFAST

PAPANICOLAOU STAIN AND RAPID ECONOMIC ACETIC

ACID PAPANICOLAOU STAIN WITH THE ROUTINE STAINS

USED IN CYTOLOGY” is a bonafide and genuine research work

carried out by me under the guidance of Dr. KUSUMA VENKATESH,

MD, DCP. Professor, Department of Pathology, Kempegowda Institute

Of Medical Sciences , Bangalore .

Date: DR.P.J. SWETHA

Place: Bangalore Postgraduate student Department of Pathology

. Kempegowda Institute of Medical Sciences,

Bangalore

III

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “COMPARATIVE

STUDY OF MODIFIED ULTRAFAST PAPANICOLAOU STAIN

AND RAPID ECONOMIC ACETIC ACID PAPANICOLAOU

STAIN WITH THE ROUTINE STAINS USED IN CYTOLOGY ” is

a bonafide research work done by DR. P. J. SWETHA in partial

fulfillment of the requirement for the degree of DOCTOR OF

MEDICINE in PATHOLOGY.

Date : DR. KUSUMA VENKATESH MD,DCP.

Place :Bangalore PROFESSOR Department of Pathology Kempegowda Institute of Medical Sciences,

Bangalore

IV

ENDORSEMENT BY HEAD OF THE DEPARTMENT

This is to certify that the dissertation entitled “COMPARATIVE

STUDY OF MODIFIED ULTRAFAST PAPANICOLAOU STAIN

AND RAPID ECONOMIC ACETIC ACID PAPANICOLAOU

STAIN WITH THE ROUTINE STAINS USED IN CYTOLOGY” is

a bonafide research work done by Dr. P. J. SWETHA under the

guidance of Dr. KUSUMA VENKATESH, MD, DCP Professor,

department of Pathology ,Kempegowda Institute of Medical Sciences,

Bangalore.

Date : DR. SUGUNA B V. MD.

Place: Professor & Head

Department of Pathology

. Kempegowda Institute of Medical Sciences,

Bangalore.

V

ENDORSEMENT BY THE DEAN AND PRINCIPAL/HEAD OF

THE INSTITUTION

This is to certify that the dissertation “COMPARATIVE STUDY

OF MODIFIED ULTRAFAST PAPANICOLAOU STAIN AND

RAPID ECONOMIC ACETIC ACID PAPANICOLAOU STAIN

WITH THE ROUTINE STAINS USED IN CYTOLOGY” is a

bonafide research work done by DR. P. J. SWETHA under the

guidance of DR. KUSUMA VENKATESH. MD, DCP, Professor,

Department of Pathology, Kempegowda Institute Of Medical Sciences,

Bangalore

Date : DR M K SUDARSHAN. MD(BHU), F.A.M.S

Place : Bangalore Dean and Principal

Kempegowda Institute of Medical Sciences

Bangalore

VI

COPYRIGHT

Declaration by the candidate

I hereby declare that the Rajiv Gandhi University of Health

Sciences, Karnataka, Bangalore, shall have the rights to preserve, use and

disseminate this dissertation in print or electronic format for academic /

research purpose.

Date: DR. P. J. SWETHA

Place: BANGALORE

© Rajiv Gandhi University of Health Sciences, Karnataka.

VII

ACKNOWLEDGEMENT

On completion of this contribution of scientific document it gives me deep

pleasure to acknowledge the guidance provided by my distinguished mentors.

With privilege and respect I like to express my gratitude and indebtedness to

my reverend,my esteemed teacher and guide, Dr. KUSUMA VENKATESH MD,

DCP,Professor , Department of Pathology, Kempegowda Institute Of Medical

Sciences,Bangalore for her constant inspiration, extensive encouragement and support,

which she rendered in pursuit of my post-graduate studies and in preparing this

dissertation right from the selection of topic till the completion, without which this

work would not have been completed.

I am grateful to my beloved teacher professor, DR. SUGUNA B. V.MD, head

of department of pathology.

I thank professors DR. RANGASWAMY MD, DNB and

DR. NIVEDITHA MD, DNB for their valuable support and guidance during my

course of study and dissertation work.

I am extremely thankful to Associate professors DR. HEMALATHA MD,

DR. SAVITHRI, DR. SUJA AJOY KUMAR, Assistant professors DR .TEJASWI

KRISHNA MURTHY, DR. SRUTHI PRASAD , DR.CHETHANA MANNEM,

DR. MANJULA and teachers DR ANAGHA JOSHI, DR. VENKATESH

PRASAD, DR. GAYATHRI, DR. PREETHA PRABHU Department of pathology,

for their valuable support and advice.

I thank Dr.M K Sudarshan, our Dean and Principal , for guidance and permitting

me to utilize resources of this esteemed institution in completion of my work.

VIII

I sincerely thank our Medical Director Dr. Capt. Venkatesh, Medical

Superintendant Dr.Suresh. I and A.M.O., Dr. Veeranna of KIMS Hospital and

Research centre for permitting me to conduct the study .

I owe my humble thanks to all lab technicians of Pathology particularly P. S. Savitha

and N. Sunandamma for their regular and timely help.

I am deeply indebted to my Parents, Parents in - Law and my husband

Dr SRIDHAR M. K. , whose constant encouragement and inspiration led me to

successful completion of my dissertation work. I would like to thank my daughter

M. S. SAANVIKA for giving me time to do my thesis work.

I express my gratitude to my senior post graduates, my co post graduates , my junior

post graduates who helped me immensely during the course of my study .

My thanks to one and all in the Library, and all hospital and college staff for their

co-operation in my study.

Last but not the least, I convey my heartfelt gratitude to all the patients,

without whose co-operation, this study would be incomplete.

Dr. P. J. SWETHA

IX

LIST OF ABBREVIATIONS

PAP - Papanicolaou

REAP - Rapid Economic Acetic Acid Papanicolaou

MUFP - Modified Ultrafast Papanicolaou

H & E - Hematoxylin & Eosin

MGG - May Grunwald Giemsa

OG - Orange G

EA - Eosin Azure

FNAC - Fine Needle Aspiration Cytology

QI - Quality Index

X

ABSTRACT

BACKGROUND AND OBJECTIVES:

Quick diagnosis of FNAC plays an important role in efficient medical

practice. The need for minimum turnaround time has encouraged newer techniques of

staining in fine needle aspiration smears, which require lesser time and are cost-

effective without compromising on the cell morphology. Needle aspiration smears has

to be assesed rapidly so that the clinicians decide on treatment options.

The objective of the present study was to to compare the results of Modified

Ultrafast Fast Papanicolaou stain(MUFP) , Rapid Economic Acetic acid Papanicolaou

stain(REAP), with Routine pap, Haematoxylin & Eosin(H&E) and May Grunwald

Giesma stain(MGG).

METHODS:

This prospective study was carried out in central laboratory, Department of

pathology, KIMS, Bangalore during December 2011 to August 2013.

Study includes 150 fine needle aspirations from lesions of organs,

thyroid(60), Breast(44), lymph nodes(36) and salivary glands(10) with patient’s

clinical details.

A minimum of 5 smears were made and stained with routine Pap, H&E,

MGG, REAP and MUFP. All smears were compared in 6 parameters and Quality

index is calculated.

XI

RESULTS:

In thyroid and lymph node PAP stain got the maximum Quality Index score

followed by H&E, REAP, MUFP and MGG. In breast PAP stain got the maximum

Quality Index score followed by H&E, REAP, MGG and MUFP. In Salivary Gland

PAP and H&E stain got the maximum Quality Index score followed by REAP, MUFP

and MGG.

CONCLUSION:

Papanicolaou stain is excellent for FNAC smears of all four organs followed

by H&E. MGG is reasonably good stain with less air drying artifacts. REAP stain is

as good as Papanicolaou stain with crisp nuclear characteristics, but it has the

disadvantage of hemorrhagic background and more air drying artifacts. Compared to

conventional Papanicolaou stain, REAP is economical and less time consuming.

MUFP stained smears showed clean background and less air drying

artifacts.In this study MUFP was found to be a good stain for inflammatory and

lymph node lesions.

Hence, REAP and MUFP can be included as routine stains in cytopathology.

KEYWORDS:

PAP, H&E, MGG, MUFP, REAP.

XII

TABLE OF CONTENTS

SL. NO. PARTICULARS PAGE NO

1 INTRODUCTION 1-2

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4-23

4 MATERIALS AND METHODS 24-32

5 OBSERVATION AND RESULTS 33-55

6 DISCUSSION 72-78

7 SUMMARY 79-80

8 CONCLUSION 81

9 BIBLIOGRAPHY 82-87

10

ANNEXURES

I.CASE PROFORMA

II.KEY TO MASTER CHART

III. MASTER CHART

88-90

91

92-93

XIII

LIST OF FIGURES

SL.NO. FIGURES PAGE

NO.

1 HASHIMOTO’S THYROIDITIS, PAP STAIN 56

2 HASHIMOTO’S THYROIDITIS, H&E STAIN 56

3 HASHIMOTO’S THYROIDITIS, HURTHLE CELL

CHANGE, H&E STAIN 57

4 HASHIMOTO’S THYROIDITIS, MGG STAIN 57

5 HASHIMOTO’S THYROIDITIS, REAP STAIN 58

6 HASHIMOTO’S THYROIDITIS, MUFP STAIN 58

7 FIBROADENOMA OF BREAST, PAP STAIN 59

8 FIBROADENOMA OF BREAST, H&E STAIN 59

9 FIBROADENOMA OF BREAST, MGG STAIN 60

10a&b FIBROADENOMA OF BREAST,REAP STAIN 60

11a&b FIBROADENOMA OF BREAST,MUFP STAIN 61

12 CARCINOMA OF BREAST, PAP STAIN 61

13a&b CARCINOMA OF BREAST, H&E STAIN 62

14a&b CARCINOMA OF BREAST, MGG STAIN 62

15 CARCINOMA OF BREAST, REAP STAIN 63

16 CARCINOMA OF BREAST, MUFP STAIN 63

17 TUBERCULOUS LYMPHADENITIS,PAP STAIN 64

18 TUBERCULOUS LYMPHADENITIS,H&E STAIN 64

19 TUBERCULOUS LYMPHADENITIS, MGG STAIN 65

XIV

20 TUBERCULOUS LYMPHADENITIS,REAP STAIN 65

21 TUBERCULOUS LYMPHADENITIS,MUFP STAIN 66

22 SQUAMOUS CELL CARCINOMA

SECONDARIES IN LYMPH NODE, PAP STAIN 66

23 SQUAMOUS CELL CARCINOMA SECONDARIES

IN LYMPH NODE, H&E STAIN 67


IN LYMPH NODE, MGG STAIN 67


IN LYMPH NODE, REAP STAIN 68


IN LYMPH NODE,MUFP STAIN 68

27 PLEOMORPHIC ADENOMAOF PAROTID, PAP

STAIN 69

28 PLEOMORPHIC ADENOMAOF PAROTID, H&E

STAIN 69

29a&b

PLEOMORPHIC ADENOMAOF PAROTID, MGG

STAIN 70

30a&b

PLEOMORPHIC ADENOMAOF PAROTID, REAP

STAIN 70

31a&b

PLEOMORPHIC ADENOMAOF PAROTID, MUFP

STAIN 71

XV

LIST OF TABLES

SL NO PARTICULARS PAGE NO.

1 AGE DISTRIBUTION OF CASES 33

2 GENDER DISTRIBUTION OF CASES 34

3 MEAN QUALITY INDEX OF FOUR ORGANS 35

4 RESULTS OF THYROID(60 CASES) 36

5 RESULTS OF BREAST(44 CASES) 38

6 RESULTS OF LYMPH NODE(36 CASES) 40

7 RESULTS OF SALIVARY GLAND(10 CASES) 42

8 H&E STAIN IN DIFFERENT ORGANS 44

9 PAP STAIN IN DIFFERENT ORGANS 45

10 MGG STAIN IN DIFFERENT ORGANS 46

11 MUFP STAIN IN DIFFERENT ORGANS 47

12 REAP STAIN IN DIFFERENT ORGANS 48

13

CORRELATION OF BACKGROUND IN H & E, PAP,

MGG, MUFP and REAP 49

14

CORRELATION OF OVERALL STAINING IN H & E,

PAP, MGG, MUFP and REAP 50

15

CORRELATION OF CELL MORPHOLOGY IN H & E,

PAP, MGG, MUFP and REAP 51

16

CORRELATION OF NUCLEAR CHARACTERISTICS

IN H & E, PAP, MGG, MUFP and REAP 52

17

CORRELATION OF CYTOPLASMIC DETAILS IN

H & E, PAP, MGG, MUFP and REAP 53

XVI

18

CORRELATION OF AIR DRYING ARTIFACTS IN


19

QUALITY INDEX IN DIFFERENT ORGANS IN

SHINDE’S STUDY AND PRESENT STUDY FOR

MUFP STAIN

73

20

QUALITY INDEX IN DIFFERENT ORGANS IN

PRIYANKA’S STUDY 74

21

QUALITY INDEX SCORES OF BREAST IN

ALMAHMOUD IDRIS STUDY AND PRESENT

STUDY

74

22

NUCLEAR FEATURES IN GUPTA’S STUDY AND

PRESENT STUDY WITH REAP 75

23

SCORING SYSTEM USED IN ASSESSMENT OF

STAINING 91

XVII

LIST OF GRAPHS

SL NO PARTICULARS PAGE NO.

1 CASE DISTRIBUTION 24

2 GENDER DISTRIBUTION OF CASES STUDIED 34

3 H&E STAIN IN DIFFERENT ORGANS 44

4 PAP STAIN IN DIFFERENT ORGANS 45

5 MGG STAIN IN DIFFERENT ORGANS 46

6 MUFP STAIN IN DIFFERENT ORGANS 47

7 REAP STAIN IN DIFFERENT ORGANS 48

8 CORRELATION OF BACKGROUND IN H & E, PAP, MGG, MUFP and REAP

49

9

CORRELATION OF OVERALL STAINING IN H & E,

PAP,MGG, MUFP and REAP 50

10

CORRELATION OF CELL MORPHOLOGY IN H & E,

PAP,MGG, MUFP and REAP 51

11

CORRELATION OF NUCLEAR CHARACTERISTICS

IN H & E, PAP,MGG, MUFP and REAP

52

12

CORRELATION OF CYTOPLASMIC DETAILS IN

H & E, PAP,MGG, MUFP and REAP 53

13

CORRELATION OF AIR DRYING ARTIFACTS IN


1

INTRODUCTION

The papanicolaou stain is a multichromatic staining technique developed by

George N Papanicolaou, the father of cytopathology in 1942 and subsequently

modified by him in 1954 and 1960.

Pap stain is used to differentiate the cells in smear preparations of various body

fluids, gynaecological smears and fine needle aspiration material from various organs.

There has been a lot of controversy as to whether wet-fixed smears stained

with Hematoxylin and Eosin or Papanicolaou stain or air-dried smears stained with

Romanowsky’s stain are better. In fact, both are complementary, but H&E and Pap

staining permit better assessment of nuclear features and are preffered by many

histopathologists.

Quick diagnosis of FNAC plays an important role in efficient medical

practice. The need for minimal turnaround time for assessing FNA smears has

encouraged innovations in staining technique that require lesser staining time with

unequivocal cell morphology.

Few rapid stains available these days include MGG stain, Diff quick stain and

toluidine blue stain. However many cytopathologists prefer the transparent,

traditional, crisp nuclear features offered by 95% ethanol fixed PAP stain rather than

air dried smears stained by Romanowsky stain.

To overcome this, ultrafast papanicolaou stain was introduced by yang and

Alvarez in 1994 which is a hybrid of romanowsky stain and pap stain. It not only

reduces the time for pap stain to 90 seconds, but also enhances the quality.

2

Pap stain requires ethanol for fixation. Ethanol is expensive and laboratory

needs a license for acquiring ethanol in bulk quantity. To overcome this REAP a less

expensive and rapid method was tried.by department of pathology of Tata Memorial

Hospital. In this method smears are prefixed in methanol and 95% ethanol baths are

replaced by 1% acetic acid.

Our aim was to search for rapid staining methods which were economical

and without compromising on the quality of cell morphology.

3

OBJECTIVES

1. To compare the results of MUFP, REAP Routine pap, Hematoxylin & Eosin

(H & E) and May Grunwald Giesma (MGG) stain.

2. To assess the quality of MUFP & REAP stain and to find the advantages over

routine stains used in cytology.

3. To find a cost-effective method which can be adapted in our laboratory.

4. To compare the quality of staining procedures used on air dried smears (MGG

and MUFP) over the wet fixed smears (Pap stain, H&E, REAP)

5. To assess the utility and applicability of these stains in cytomorphological

study in FNAC of thyroid, breast, lymph nodes and salivary gland lesions.

4

REVIEW OF LITERATURE

Cytology started as a revolutionary idea of looking at imprints of cut tumors

surface at postmortem. It has evolved through many new methods of procuring, fixing

and staining cells. Its main attribute lies in its ability to allow prompt, accurate

assessment of cell changes on material taken with minimally invasive procedure and

processing.1

The evolution of cytopathology occurred through four overlapping eras; the

early history (1860-1940); the development and expansion of exfoliative cytology in

the USA and elsewhere (1940-1960); the consolidation of cytopathology as a

discipline and the parallel developments of population screening and FNA cytology

(1955-1985); the maturation of cytopathology as a discipline and its integration with

new technology (1985 to the present day).1

In the early historical era microscopic observations of normal and abnormal

human cells either in exfoliated or in imprints or scrapes were steadily and

independently recorded throughout the nineteenth century.2, 3. By first decade of

twentieth century, exfoliative cancer cells had been described in all types of

specimens.4

In 1922, professor LS Dudgeon used cytology at St. Thomas Hospital for the

diagnosis of a wide variety of neoplastic and inflammatory diseases from imprints of

surgical specimens5. Dudgeon considered that the stained films were much nicer to

examine than paraffin sections.6 At the same time in USA, FNA cytology was being

developed and the first series on aspiration of neoplasms was published from

memorial Hospital for cancer and allied diseases in New York City.7

5

A second era of cytopathology began in 1941 with the publication of an the

diagnostic value of vaginal smears in carcinoma of uterus by George N. Papanicolaou,

an anatomist, and Herbert F. Traut, a gynaecologist.8 Papanicolaou’ contribution to

this field was two-fold; he recognized the importance of wet fixation of cytological

specimens and he systematically began to accumulate examples of cancer cells in

vaginal smears, culminating in his paper New Cancer Diagnosis.9

Concurrently with the development of cervical screening the cytological

method of cancer diagnosis began to be more widely applied to the respiratory,

alimentary and urinary tracts as well as to the serous cavities and the central nervous

system.1

The era of consolidation was heralded by two publications: the first issue of

Acta cytologica in 1957, the oldest journal devoted exclusively to cytopathology; and

in 1961, by the publication of Diagnostic cytology and its Histopathologic bases by

Leopold G.Koss in association with Grace R. Durfee.10 In the last 60 years there is an

explosion in the literature of cytopathology, with thousands of articles and scores of

books written on the subject.1

Population based cervical screening is now practiced to a greater or lesser

extent in almost all countries of the developed world.1 The imperial cancer Research

Fund Coordinating Committee on cervical screening made the statement in 1984 that

‘with the exception of stopping smoking, cervical cytology screening offers the only

major proved public health measure for significantly reducing the burden of disease11,

its introduction was highly controversial and has remained so at every stage of its

development. By 1986, there was sufficient evidence from an international

multicentre analysis to show that 5 yearly and 3-yearly screening reduced the risk of

6

invasive cancer by 84% and 93% respectively, while little additional benefit was

achieved by annual screening.12

The impetus of the development of cytopathology as we know it today

resulted from the painstaking research of papanicolaou in the USA. Thus

papanicolaou is justly referred to as the ‘Father of cytopathology’.13

PAPANICOLAOU STAIN :

For the routine diagnostic cytology, the papanicolaou stain is recommended.

The use of the papanicolaou stain results in well-stained nuclear chromatin,

differential cytoplsmic counterstaining, and cytoplasmic transparency.14 Although,

originally developed for imterpretation for gynaecological specimens, it is now

commonly used to facilitate the accurate detection and interpretation of abnormal

cells from variety of sources.

NUCLEAR STAINS 15 :

Haematoxylin - It is extracted from the logwood of the tree Haematoxylon

campechianum. Oxidation of this extract produces a coloured substance ‘haematein’,

which itself is a poor dye, but in the presence of a metallic mordant, forms a most

powerful stain.

Natural oxidation involves exposure of solutions of hematoxylin to sunlight

and air. Chemical oxidation is achieved by the addition of oxidizing gents such as

mercuric oxide, sodium iodate and Potassium permanganate. The metallic mordants

used either Alum mordants or Iron.

7

It is used as progressive or Regressive staining. In progressive staining the

reaction is stopped once the desired staining intensity is achieved. In Regressive

staining, longer time is required to over stain the tissue before the stain is selectively

removed in acid alcohol (1% hydrochloric acid in 70% ethanol).

CYTOPLASMIC STAINS 15:

There are multiple acid dyes that provide differential counterstaining and

cytoplasmic transparency.

Organe G6 (OG 6).

Eosin Ozure 36 (EA 36 or EA 50) which contains light green, Eosin and

bismark brown.

MODIFICATION OF PAPANICOLAOU STAIN : 14

Modification of the original papanicolaou stain (1942) was published by Dr.

Papanicolaou in 1954 and 1960. Papanicolaou Technique 1 uses Harris hematoxylin

regressively. Papanicolaou Technique II, described for urinary and gastric

preparations, uses hematoxylin progressively. Other modifications include Gill’s

modification, Miller’s modification, Saccomanno’s modification for carbowax fixed

smears and Durfee’s modification for urine sediment smears.

FIXATIVES: 14

Rapid fixation of smears is necessary to preserve cytological details of cells.

For many years the fixative of choice for gynecologic and other smear

preparations was the one recommended by Papanicolaou, a solution of equal parts of

8

ether and 95% ethyl alcohol. Subsequently, it is abandoned because ether presents a

fire hazard. Ninety-five percent ethyl alcohol (ethanol) is now employed as a fixative

by many laboratories, with excellent results. Smears should remain in 95% ethanol

fixative for a minimum of 15 minutes prior to staining.

To obtain ethanol without federal taxation, a license is required. However, to

obtain results similar to those seen with 95% ethonal, different concentrations must be

used.

EQUIVALENT CONCENTRATIONS OF SEVERAL ALCOHOLS FOR

PURPOSES OF CELL FIXATION

100% Methonal

95% Ethanol

95% Denatured alcohol

80% proponal

80% Isopropanol

WET FIXATION : 14, 16

Wet fixation is traditional method of fixation in which smears are immediately

kept in fixative before air-drying of smears. The disadvantages of wet fixed smears

are air-drying artifacts, hemorrhagic background and cell loss during fixation.

To overcome these disadvantages, in 1988, Chan and Kung reported that air-

dried smears can be rehydrated by immersing the smears in 0.9% Nacl for 30sec

which is used in MUFP.

9

FINE NEEDLE ASPIRATION CYTOLOGY :

FNA was first introduced in Sweden by Franzen, a haematologist & oncologist

by training, who used the same Romanowsky staining method as for bone marrow

aspirates.17 The technique was further developed by Soderstrom, Fox and also by

Lopes Cardoso, Von Haam, Crepinko and Hauptmann.18 In the UK, FNA was

pioneered by a surgeon, John webb, who was given enthusiastic support by some of

the renowed cytopathologists of the time.19 The technique also became popular in the

USA after a long interval since its early use in the 1930s.

The present day focus of FNA cytology is on obtaining a satisfactory

specimen on which a reliable diagnosis can be made and therefore, that the aspirate

sample should provide true reflection of the disease process.20 FNA is now

established as the first line investigation of mass lesions whereever they occur in the

body.20

ADVANTAGES OF FNA : 20

- The technique is relatively painless, produces a speedy result and is

economical.

- The method is applicable to lesions that are easily palpable. New

radiological techniques for internal imaging of organs and lesions opened

the door for FNA of deeper impalpable structures.

- Costly days in hospital can be avoided as tissue diagnosis may be

obtained within minutes rather than days as FNAC can be done in OPD.

10

- The low risk of complications is an additional advantage that allows FNA

cytology to be performed as an office procedure.

- Cells obtained by FNA can be manipulated in a variety of ways useful to

research: for DNA analysis (polymerase chain reaction), ultra structural

study, Immunocytochemistry, gene rearrangement, morphometry and

image analysis.

LIMITATIONS OF FNAC :

FNA cytology has its limitations. Sampling can be scanty, and histological

architecture is lost thereby rendering diagnosis of lesions difficult based on

morphology.20

There is a risk of complications of FNA. The overall morbidity and mortality

to FNA has been estimated in several studies and the risk of death is approximately 1

in 1500. Serious complications have been reported such as major hemorrhage after

FNA of lung, liver and kidney; septicimea after prostate aspiration; bile peritonitis

following needling of liver; and acute peritonitis resulting from pancreatic aspiration.

However, such complications are very rare. Review of literature shows that multiple

passes, larger needles, and absence of normal parenchyma covering the lesion appear

to increase the risk.20 ‘

Contraindications to deep site aspirates include anticoagulant therapy and

intrinsic bleeding problems as they increase the risk of bruising and hemorrhage.

Intractable cough and poor respiratory function are absolute contraindications to

transthoracic FNA. Aspirates of unsuspected hydatid cyst carry the potential risk of

11

anaphylactic shock resulting from rupture and is best avoided. FNA of

phaeochromocytomas is contraindicated for fear of inducing a hypertensive crisis.20

NEED FOR RAPID ASSESSMENT IN CYTOLOGY :

Quick diagnosis of fine-needle aspiration cytology (FNAC) plays an important

role in efficient medical practice.22 Rapid assessment of FNA smears has become

increasingly popular due to the global trend in reducing health care costs. The goal is

minimal time for hospitalization and the fastest possible turnaround time for test

results.23 Immediate examination of the aspirates for adequacy, while the patient

remains in the biopsy suite, reduces the number of inadequate samples and decreases

the number of needle passes performed.24

The need for minimal turnaround time for assessing fine needle aspiration

smears has encouraged innovations in staining technique that require lesser staining

time with unequivocal cell morphology.25

Two fundamentally different methods are used for routine fixation and

staining of cytologic specimens. Romanowsky-type stains (e.g., Wright’s, May-

Grunwald Giemsa, Diff-Quick) are based on air-drying. The trichrome papanicolaou

and bichrome Hematoxylin and Eosin are based on wet-fixation.26

Many cytopathologists prefer the transparent, traditional, crisp nuclear features

offered by 95%, ethanol fixed papanicolaou stained cytology specimens to the opacity

of nuclei, nuclear enlargement, and flatness of image in air-dried smears stained by

Romanowsky stains.25

12

MODIFIED ULTRAFAST PAPANICOLAOU STAIN (MUFP) :

To overcome the disadvantages of both Romanowsky and pap stains, Yang

and Alverez in 1995 suggested an ultrafast Papanicolaou (UFP) stain, which is a

hybrid of Romonwsky and Pap stains, and requires only 90 seconds. It involves

rehydration of air dried smears, fixation in alcoholic formalin and subsequent pap

staining except that the duration of each step is shortend.25

ULTRAFAST PAPANICOLAOU STAIN 23

1. Normal saline 30 seconds

2. 95% Ethanol (optional), for

Storage/transport

3. Alcoholic formalin 10 seconds

4. Water 6 slow dips

5. Richard –Allan Hematoxylin 2 2 slow dips

6. Water 6 slow dips

7. 95% Ethanol 6 slow dips

8. Richard-Allan Cytostain 4 slow dips



11. Xylene 10 slow dips

Mount and coverslip

Earlier rapid papanicolaous stains (Kline’s rapid and Tao’s rapid) are identical

to the routine stain except that the duration of each step is shortend. The problem with

rapid papanicolaou stain is fourfold : (1) Both the cytoplasm and nucleus lose much

cellular detail from inadequate fixation ;(2) Since FNA samples are inherently bloody,

the tumor cells are often covered with ubiquitous RBC’s this is particularly annoying

with papanicolaou stain as RBC’s stain orange; (3) The wet-fixed cells are much

smaller than air-dried cells; and (4) Loss of wet fixed cells during processing.23

13

To overcome the first problem, fixative is changed from 95% ethanol to 4%

formaldehyde in 65% formalin.23 Alcoholic formalin differentiates RNA from DNA

in subsequent staining because of acidic PH (PH-5). It renders the nucleoli red and

colours more vibrant.25

The last three problems can be overcome by chan and kung’s rehydration of

air-dried smears. Air drying allows the cells to stick firmly to the glass slide and the

rehydration in normal saline allows RBCs to hemolyse, unmasking the cells for

morphologic analysis.23

The chief limitation of ultrafast papanicolaou stain, is that Richard Allan

Haematoxylin (RA-H) and Richard Allan cytostain (RA-C), used in the staining

procedure are not universally available.25

MM Kamal and MM Munshi (2000) made two modifications in the ultrafast

pap stain. First, Instead of Richard Allan Haeatoxlyin (RA-H), Gill’s Haematoxlyin is

used. Second modification was instead of Richard Allan cytostain (RA-C) which is an

alcoholicmixture of orange G, Eosin Y, Light Green and Aniline blue, they used EA

modified which is an alcoholic mixture of Eosin Y, light green, Phosphotungstic acid

and glacial acetic acid.25

As orange G was omitted from the staining solution in Modified ultrafast pap

stain, orange discoloration was no longer a problem. Modified ultrafast papanicolaou

stain can be used for tissues where chances of cytoplasmic keratinization is negligible.

14

MODIFIED ULTRAFAST PAPANICOLAOU STAIN :

0.9% Normal saline (30sec)

Alcoholic formalin (10 sec)

6 dips water

Gill’s Haematoxylin (30 sec)

6 dips water

95% Alcohol (6 dips)

EA Modified (15 sec)

95% Alcohol (6 dips)

100% Alcool (6 dips)

Xylene (6 dips)

Modified ultrafast papanicolaou (MUFP) is useful in rapid assessment of

adequacy of smears and for intra operative FNA consultations. 25, 27

Priyanka and Sudhamani et al. showed that Harri’s hematoxylin gives good

staining as much as Gill’s hematoxylin in MUFP.33

15

A study was conducted by Junko Maruta and Hironobu Hashimoto et al., in

2002 showed the applicability of modified ultrafast stain for quick diagnosis of

thyroid diseases. Two specimens from each of 251 thyroid aspirations (122 malignant

and 131 benign) were prepared using the modified ultrafast stain and the standard

papanicolaou stain. The sensitivities of cytologic diagnosis in specimens stained by

standard papanicolaou method and the modified ultrafast method were 95.0% and

93.3% respectively, and the specifities were 99.2% and 97.7% respectively.22

Another study done by Grace C.H.Yang and Doreen et all in 2001 on

ultrasound guided FNA of thyroid showed that MUFP by high lighting the ”orphan

Annie-eyed” clear nuclei, help to differentiate follicular variant of papillary thyroid

carcinoma from follicular neoplasms.28

MM Kamal and MM Munsi et al., done a stdy on ‘Efficacy of modified

ultrafast papanicolaou stain for Breast aspirates’ in 2000. In this study smears from

FNA from 100 breasts lumps were stained by the MUFP stain. Eighty six breast

aspirates are adequate for interpretation. Smears showed transparent cells with crisp

nuclear features, equal to and even better than the conventional papanicolaou stain, in

a blood free background.25

Shinde and Ajita et al., done a study on ‘Application of Modified ultrafast

papanicolaou stain in cytology of various organs’ in 2005. In their study, Group-I

included 40 FNAC smears of various organs. In each case, three smears were

prepared and stained by MUFP, Papanicolaou, and MGG stains. In groups II, 10

intraoperative cytology smears of different organs were sained with MUFP and rapid

Haematoxylin – Eosin(H & E). For assessment of MUFP stain, scores were given on

four parameters; background of smears, overall staining pattern, cell morphology and

16

nuclear staining. Quality index was calculated from ratio of score achieved to

maximum possible score. Diagnosis made by MUFP stain was compared with

standard stains. The diagnosis was correct except in three cases of metastatic

squamous cell carcinoma. Hence, it was concluded that MUFP stain is useful for

rapid diagnosis by FNAC, but is not useful for squamous cell lesions.29 This is

because Orange G is not being used in this method.

Grace C.H.Yang and Jerry Waisman done a study on Salivary Fine-needle

aspirates in 2005. They compared 20 cases of Adenoid cystic carcinoma to 15 cases

of Cylindromatous pleomorphic adenoma and 9 cases of basal cell adeoma. All

smears are stained with diff-quick, hematoxylin and eosin, Papanicolaou or ultrafast

papanicolaou stain. The study concluded that Adenoid cystic carcinoma can be

distinguished from cylindromatous pleomorphic adenoma and basal cell adenoma

using ultrafast pap stain and oil immersion lens by revealing a difference in the

nuclear features and the difference in the amount of cytoplasm.30

Luciano.B Lemos and Mithra Baliga in 1997 did a one year study in Fine

Needle Aspiration and concluded that ultrafast papanicolaou stain is particularly

useful in diagnosing squamous carcinoma because of the bright orange staining it

imparts to keratinizing squamous carcinoma cells, which is an important

consideration in the diagnosis of many head and neck carcinomas as well as

metastatic carcinomas.31

Kenji Bando and Reiji Haba et al., done a study on ‘Utility of Immediate

cytologic Diagnosis of Lung masses using ultrafast papanicolaou stain’ in 2011. In

this study out of 503 cases investigated, the results of immediate cytology using

ultrafast pap stain were positive in 348 cases and negative in 153 cases. The study

17

concluded that immediate cytology can be implemented fairly easily in any hospital,

and is superior technique for obtaining high diagnostic accuracy.32

Priyanka Choudhary and Sudhamani S et al., did a study on ‘Comparison of

MUFP with the standard rapid papanicolaou stain in cytology of various organs’ in

2012. In this study a total of 100 FNAC cases were studied by random sampling. Two

smears were prepared for each case and stained by both the MUFP and the rapid pap

stain. Scores were given and the quality index was calculated, followed by the

statistical analysis. The cases included lymphnode (43), thyroid (25), breast (23),

salivary gland (02), and soft tissues (07). Scores were given on four parameters :

Background of smears, overall staining pattern, cell morphology and nuclear staining.

Quality index was calculated from the ratio of score achieved to the maximum score

possible. The study concluded that quality index of MUFP smears was better

compared to the rapid pap stain in all the organs, and was statically significant.33

Another study was conducted by Shuji Bandoh and Jiro Fujita et al., in 2013

on ‘Diagnostic Accuracy and safety of Flexible Bronchoscopy with Multiplanar

Reconstruction images(MPR) and Ultrafast Papanicolaou Stain (UFP). This study

includes one hundred consecutive patients with solitary pulmonary nodule who

underwent bronchoscopy with multiplanar reconstruction and MUFP stain. The total

diagnostic accuracy of bronchoscopy in the MPR and UFP group (91%) was

significantly higher compared with the historical control group(58%) [P<0.05]. The

conclusion of the study was that combined use of MPR image and UFP during

flexible bronchoscopy improved diagnostic accuracy and safety in evaluating solitary

pulmonary nodules.34

18

M.Kamal and Madhura M.Kulkarni et al., in 2011 did a study to find out the

efficacy of the ultrafast papanicolaou staining technique for immediate cytologic

diagnosis and to check specimen adequacy during radiologically guided FNAC

procedure. In this study Group I included 238 out patient FNACs, groups II included

59 radiologically guided FNACs and group III included 50 cases of intraoperative

cytology. Overall diagnosis was possible in 297(85.6%) cases. Only 8 (2.3%) cases

could not be diagnosed due to staining difficulties. The overall concordance rate was

98%. The conclusion of the study was UFP staining technique is an accurate and

reliable method for rapid cytology reporting. It significantly reduces total turnaround

time of the test result, thereby it is cost-effective both for the patient and the

hospital.35

RAPID ECONOMIC, ACETIC ACID, PAPANICOLAOU STAIN : (REAP)

The universal stain for cervical cytological screening is papanicolaou stain. It

yields a polychromatic, transparent staining reaction with crisp nuclear and

cytological features. However it utilizes a considerable amount of ethyl alcohol and

takes about 20 minutes. In rapid pap technique, staining is achieved in 90 seconds but

it requires a substantial volume of ethanol which is expensive . In India a laboratory

needs a license for acquiring ethanol in bulk quantity, obtaining a license and its

renewal is a difficult task.36

To overcome this Department of Pathology, Tata Memorial Hospital, Mumbai

introduced a modified technique referred as ‘Rapid, Economic, Acetic acid

Papanicolaou stain’ (REAP). It is rapid and as economical as standard Papanicolaou

stain without compromising staining quality.37

19

RAPID, ECONOMIC, ACETICACID PAPANICOLAOU STAIN:

Only in the first step for fixation and in last sep for dehydration, absolute

alcohol was used which is same as standard Papanicolaou stain.

Method for REAP stain :

1% acetic acid 10 dips

Harris Haematoxylin , preheated 600 C 10 dips

Tap Water 10 dips


OG-6 10 dips


EA-50 10 dips


Methanol 10 dips

Xylene 10 dips

Blotting was done after each step.

Mount by D.P.X

The ethyl alcohol grades used in conventional PAP stain are replaced by 1%

acetic acid.36 Acetic acid is mild dehydrating agent. The main advantages of using

acetic acid in place of alcohol are its easy availability and extremely low cost.38 Harris

hematoxylin is used in both methods for nuclear staining but the time is 1 minute in

conventional PAP stain and in REAP it is reduced to 10 dips as the stain is preheated

to 600C. Heating of hematoxylin is done in water bath at 600C before staining for

rapid penetration. In standard PAP stain, the blueing agent is Scott’s tap water which

20

is replaced by ordinary tap water in REAP stain. The cytoplasmic stains OG 6 and

EA 50 are same in both methods except the time spent. In REAP the smears are

washed in 1% acetic acid and final dehydration is by methonal (10dips). Clearing is

done by single change of xylene (10 dips) in REAP.36

Dighe and Duthan Ajit et al., did a study in 2005. In this study smears from

200 patients were collected and fixed in methanol. Half were subjected to

conventional papanicolaou and half were stained with REAP stain. With REAP

method, cytoplasmic and nuclear staining was optimal in 181 and 192 cases,

respectively. The staining time was considerably reduced, to 3 minutes and the cost

per smear was reduced to one fourth. The staining quality remained good in all the

smears for >2 years. The study concluded that REAP is a rapid, cost-effective

alternative to Papanicolaou stain. Though low stain penetration in large clusters is a

limitation, final interpretation was not compromised.37

Ranu Roy Biswas and chandi.C. Paral et al., did a study in 2008. 220 PAP

smears from 110 patients (2 per subject) were collected. One set of smears was

stained by conventional PAP stain and the other set by REAP stain. In REAP

technique , cytoplasmic and nuclear staining was optimal in 100 and 105 cases

respectively. The cost was reduced to 25% due to limited alcohol use and the staining

time was minimized to 3 minutes. This study concluded that REAP stain, in

comparison to conventional papanicolaou, provides a suitable, excellent and rapid

alternative for cytological screening with minimum cost. The stain preservation is

also good in REAP method.36

S.Gupta and K.L.Chachra et al, did a study in 2009. Five hundred paired

cervical smears were collected from women as part of routine cervical cancer

21

screening. One set of smears was stained by conventional pap staining protocol and

the other by modified protocol in which alcohol was replaced by 1% acetic acid in all

the steps except during fixation and prior to mounting. In addition one alcohol-based

counter stain, OG, was omitted. The study concluded that improvised pap staining

protocol with minimum alcohol use is a simple, cost-effective and technician-friendly

procedure that can be easily adopted in high-volume, resource-limited Laboratories

for mass cervical cancer screening.38

Advantages of Rehydration of Air-dried smears over Wet Fixed smears :

There has been a lot of controversy as to whether wet-fixed smears stained

with H & E or PAP stain or Air-dried smears stained with Romanowsky’s stain are

better. In fact, both are complementary, but H & E and PAP staining permit better

assessment of nuclear features and are preferred by many histopathologists.40

However, staining with H & E and PAP becomes highly unsatisfactory once air

drying has occurred.41,42. Drying artifacts are difficult to avoid, because it takes time,

no matter how minimal, to spread the aspirated material and transfer the slide to the

fixative.39

The various redehydrating agents, that have been described by various

researchers include.

- Tap water

- Normal saline.39

- 50% Aqueous glycerin.43

- Acetic acid- alcohol solution.44

22

- Hydroxypropyl methyl cellulose ether in water.

Of these normal saline was found to give the best results. The nuclear

chromatin and nucleoli were as crisp as those in the wet-fixed smears, and the

cytoplasmic outlines remained distinct.39

The method of Rehydration offers several advantages over wet fixation. First,

the smears can be spread more thinly and leisurely. Second, the problem of air drying

in the edges of the smear can be avoided.39 Third, when a wet smear is placed in 95%

ethanol the larger particles or thicker portions of the smear may fall of.45 If the

smears are fully air-dried, the cells adhere better to the slide. Fourth, lysis of most of

the red blood cells creates a much less distracting background to permit better

cytologic assessment. Fifth, the cells appear flatter and the depth of focus on the

nuclei is much more shallow. Sixth, to make best use of all materials available, the

slide used for spreading the smears may also be salvaged by rehydration for cytologic

examination.39

John K.C.Chan and Ignatius.T.M.Kung et al., did a study on ‘Rehydration of

Air-Dried smears with Normal saline’ in 1988. In this study air-dried smears are

placed in normal saline for 30 seconds before fixation in 95% alcohol. They found

that optimal time for rehydration of air-dried smears in normal saline range from 5

seconds to 5 minutes and best results were obtained if air drying did not exceed 30

minutes.39

Wai.F.Ng and Fook B, Choi et al., did a study in 1994. In this study, ninety

fluid specimens (30 cases of urine, ascitic and pleural fluid) were studied by preparing

three comparable smears. One was air dried for Giemsa stain, one wet fixed in 95%

23

ethanol and one dried on a hot plate at 370C, rehydrated in normal saline for 30

seconds and fixed in ethanol. The latter two were stained with papanicolaou stain, and

a comparision was made about retention of red blood cells, retention of epithelial or

mesothelial cells and cytologic preparation. The study concluded that the rehydrated

smears showed a decrease in the chromaticity of staining, more flattened cell clusters

and slight cell enlargement. The rehydration method was beneficial for urine and

blood-stained body cavity fluids.46

C.A.Jones did a study on ‘Papanicolaou staining of air-dried smears : value in

rapid diagnosis’ in 1996. The study concluded that air-dried material stained after

rehydration showed superior nuclear definition compared with wet-fixed material and

the removal of erythrocytes enhanced the staining of the remaining epithelial cells.47

24

MATERIALS AND METHODS

This prospective study was carried out in central laboratory, Department of pathology,

KIMS, Bangalore during December 2011 to August 2013.

Study includes fine needle aspiration from lesions of organs i.e., thyroid, breast,

lymph nodes and salivary glands with patients clinical details.

Total number of cases studied - 150

Thyroid - 60

Breast - 44

Lymph node - 36

Salivary gland - 10

GRAPH 1. Case distribution:

Thyroid

Breast

Lymph node

Salivary gland

CASES

25

PROCEDURE FOR SMEAR PREPARATION AND FIXATION:

The FNA procedure was performed in our laboratory by standard method. A total

of minimum 5 smears were made on clean glass slides of which 2 smears were fixed

in 95% ethanol for a minimum of 15 minutes. These smears were submitted for pap

stain and H & E stain.

One smear was fixed in methanol and submitted for REAP stain. The remaining

2 smears were air dried out of which one was stained by MGG stain and other smear

was rehydrated with normal saline and subsequently fixed in alcoholic formalin and

stained by MUFP stain.

Inclusion Criteria:

Fine needle aspirations from thyroid,breast,salivary gland and lymph node

lesions done in central laboratory, Department of Pathology, KIMS, Bangalore.

Exclusion Criteria:

Fine needle aspirations from lesions of other organs, Cervico-Vaginal smears

and smears from body fluids were excluded.

26

STAINING PROCEDURES OF PAP, H&E, MGG, REAP AND MUFP

PAPANICOLAOU METHOD

REAGENTS REQUIRED :

1. Harri’s Haematoxylin

(Without acetic acid)

2. Orange G 6 (OG 6).

0.5 Orange G in 95% alcohol 100 ml

Phosphotungstic acid 0.15g.

3. Eosin azure 36 (EA 36 OR EA 50)

0.5 Light green SF yellow in 95% alcohol 45ml

0.5% Bismark brown in 95% alcohol 10 ml

0.5% Eosin Y in 95% alcohol 45 ml

Phosphotungstic acid 0.2 g

Saturated aqueous lithium carbonate 1 drop

TECHNIQUE :

1. Fix smears (while still moist) in 95% alcohol – 15 mnts.

2. Rinse smears in distilled water.

3. Stain in Harri’s haematoxylin for 4 mnts.

4. Wash in tap water for 1-2 mnts.

5. Differentiate in acid alcohol (25% HCL in 70% alcohol).

6. Blue in tap water or 1.5% sodium bicarbonate.

7. Rinse in distilled water.

8. Transfer to 70% alcohol, then 95% alcohol for a few seconds.

9. Stain in O G6 for 1-2 minutes.

10. Rinse in 3 changes of 95% alcohol for a few seconds.

11. Stain in EA 50 for 3 – 5 minutes.

12. Rinse in 3 changes of 95% alcohol for a few seconds.

27

HAEMATOXYLIN AND EOSIN STAIN

Reagents required :

1. Harris Haematoxylin

2. 1% Eosin.

3. 1% Acid alcohol.

Technique :

1. Fix smear in 95% alcohol – 15 min

2. Wash with water.

3. Stain with Harris Haematoxylin – 5 minutes.

4. Wash with water.

5. Dip in 1% Acid alcohol.

6. Wash in running tap water until blueing.

7. Counter stain with 1% Eosin.

Dehydration :

1. Rinse in 70% Alcohol for 30 seconds.

2. Rinse in 90% Alcohol for 90 seconds.

3. Rinse in Absolute Alcohol for 30 seconds.

4. Rinse in two changes of Xylene.

5. Clear and mount with D.P.X.

28

MAY GRUNWALD GIEMSA STAIN(MGG)

Stock Solution Of MGG:

0.5 grams of MGG powder dissolved in 100 ml of methanol.

Stock Solution Of Giemsa:

0.75 grams of Giemsa Powder dissolved in 100 ml of methanol.

Working Solution Of MGG:

Two parts of Stock Solution Of MGG and one part of methanol.

Working Solution Of Giemsa:

One part of Stock Solution of Giemsa and nine parts of distilled water.

Staining Technique:

Stain with Working Solution of MGG in 1-2 minutes.

Dilution with Working Solution of Giemsa 10 minutes and wash in tap water and dry.

29

REAGENTS USED IN REAP STAIN:

1.Methanol

2.Tap water

3.Harris Hematoxylin

4.Orange G- 6

5.EA-50

6.1% Acetic acid

7.Xylene

8.DPX

REAGENTS USED IN MUFP STAIN:

1. Normal Saline

2. Alcoholic Formalin- 3 liters of alcoholic formalin is prepared by following (pH 5)

300 ml of 40% Formalin

2053 ml of 95% Alcohol

647 ml of Distilled water

3. Gill’s Hematoxylin

4. 95% Alcohol

5. 100% Alcohol

6. Tap water

7. EA- 50

8. Xylene

30

RAPID, ECONOMIC, ACETIC AND PAP STAIN (REAP)

Methanol (30 sec )

1% acetic acid (10 dips )

Harris hematoxylin, preheated to 60◦C (10 dips)

Tap water (10 dips)

Acetic Acid (10

OG -6 (10 dips)

1% acetic acid (10 dips)

EA-50 (10 dips)

1% Acetic acid (10dips)

Methanol (10 dips)

Xylene (10 dips)

31

MODIFIED ULTRAFAST PAPANICOLAOU STAIN (MUFP)

Air dried smears

Normal saline (30 sec) hydration

with in 30 min

Alcoholic Formalin (10 secs)

Tap water (6 dips)

1% Gills hematoxylin (30 secs)

Tap water (6 dips)

95% Ethanol (6 dips)

EA-50 ,15 sec (4 dips)

100% alcohol (6 dips)

Xylene (10 dips)

95% Ethanol (6 dips)

32

SCORING SYSTEM USED IN ASSESSMENT OF STAINING:

PARAMETER SCORE=1 SCORE=2 SCORE=3

BACKGROUND HEMORRHAGE CLEAN

OVERALL STAINING POOR AVERAGE GOOD

CELL MORPHOLOGY POORLY

PRESERVED

MODERATELY

PRESERVED

WELL

PRESERVED

NUCLEAR

CHARACTERISTICS

SMUDGY

CHROMATIN

MODERATELY

CRISP

CHROMATIN

CRISP

CHROMATIN

CYTOPLASMIC DETAILS UNSATISFACTORY SUBOPTIMAL OPTIMAL

AIR DRYING ARTIFACTS >50% <50% 0%

The maximum score for a single case, taking into account all the six parameters,

was 17.

The “Quality Index” was obtained by calculating the ratio of actual score obtained

to the maximum score possible.

Quality Index= actual score obtained /maximum score possible

Quality Index for each of the five stains of the four organs was compared.

33

OBSERVATION AND RESULTS

TABLE NO 1

Age distribution of cases studied

Age in

years Thyroid

Lymph

Node Breast

Salivary

Gland Total

1-10 1(1.7%) 0(0%) 0(0%) 0(0%) 1(0.7%)

11-20 1(1.7%) 4(11.1%) 0(0%) 0(0%) 5(3.3%)

21-30 14(23.3%) 3(8.3%) 8(18.2%) 0(0%) 25(16.7%)

31-40 24(40%) 11(30.6%) 12(27.3%) 3(30%) 50(33.3%)

41-50 9(15%) 9(25%) 19(43.2%) 2(20%) 39(26%)

51-60 7(11.7%) 5(13.9%) 3(6.8%) 3(30%) 18(12%)

61-70 2(3.3%) 3(8.3%) 2(4.5%) 1(10%) 8(5.3%)

71-80 2(3.3%) 1(2.8%) 0(0%) 1(10%) 4(2.7%)

Total 60(100%) 36(100%) 44(100%) 10(100%) 150(100%)

Mean ±

SD 39.17±13.82 42.92±14.69 41.41±10.56 50.70±13.69 41.49±13.35

Distribution of cases in thyroid is maximum in age group of 31-40 (40%) and 21-30

(23.3%) years.

Distribution of cases in lymph node is maximum in age group of 31-40 (30.6%) and

41-50 (25%) years.

Distribution of cases in breast is maximum in age group of 41-50 (43.2%) and 31-40

(27.3%) years.

Distribution of cases in salivary gland is maximum in age group of 31-40 (30%) and

51-60 (30%) years.

Gender distribution of cases studied

Gender Thyroid

Male 4(6.7%) 21(58.3%)

Female 56(93.3%) 15(41.7%)

Total 60(100%) 36(100%)

P=<0.001 by Fisher Exact test

Gender distribution of patients is significant.

Thyroid lesions are more common in females (93.3%).

Lymph Node lesions are common in males (58.3%).

Breast lesions are common in females (97.7%).

Salivary gland lesions are common in males (60%).

GRAPH 2. Gende

0

10

20

30

40

50

60

70

80

90

100

Thyroid Lymph Node

Per

cen

tag

e

TABLE NO 2

Gender distribution of cases studied

Lymph

Node Breast

Salivary

Gland Total

21(58.3%) 1(2.3%) 6(60%) 32(21.3%)

15(41.7%) 43(97.7%) 4(40%) 118(78.7%)

36(100%) 44(100%) 10(100%) 150(100%)

P=<0.001 by Fisher Exact test

Gender distribution of patients is significant.

Thyroid lesions are more common in females (93.3%).

Lymph Node lesions are common in males (58.3%).

Breast lesions are common in females (97.7%).

and lesions are common in males (60%).

GRAPH 2. Gender distribution of cases studied

Lymph Node Breast Salivary Gland

Female Male

Gender

34

Total

32(21.3%)

118(78.7%)

150(100%)

35

TABLE NO 3

MEAN QUALITY INDEX OF FOUR ORGANS

Quality index were calculated for all the slides of each organ and mean was

calculated.

In Thyroid PAP stain got the maximum Quality Index score followed by H&E,

REAP, MUFP and MGG.

In Breast PAP stain got the maximum Quality Index score followed by H&E, REAP,

MGG and MUFP.

In Lymph Node PAP stain got the maximum Quality Index score followed by H&E,

REAP, MUFP and MGG.

In Salivary Gland PAP and H&E stain got the maximum Quality Index score

followed by REAP, MUFP and MGG.

Before calculating mean statistics were applied first to specific organ then inter organ

comparision was calculated.

CASES H&E PAP MGG MUFP REAP

THYROID 60 0.94 0.96 0.81 0.82 0.85

BREAST 44 0.94 0.96 0.81 0.80 0.92

LYMPH NODE 36 0.92 0.95 0.81 0.85 0.88

SALIVARY

GLAND

10 0.93 0.93 0.80 0.82 0.85

36

TABLE NO 4

THYROID (60 CASES)

H&E PAP MGG MUFP REAP

Background

• Hemorrhagic 23(38.3%) 18(30%) 49(81.7%) 14(23.3%) 44(73.3%)

• Clean 37(61.7%) 42(70%) 11(18.3%) 46(76.7%) 16(26.7%)

Overall staining

• Poor 0(0%) 0(0%) 2(3.3%) 0(0%) 0(0%)

• Average 2(3.3%) 1(1.7%) 39(65%) 36(60%) 24(40%)

• Good 58(96.7%) 59(98.3%) 19(31.7%) 24(40%) 36(60%)

Cell morphology

• Poorly preserved 0(0%) 0(0%) 1(1.7%) 1(1.7%) 0(0%)

• Moderately

preserved 4(6.7%) 1(1.7%) 19(31.7%) 31(51.7%) 16(26.7%)

• Well preserved 56(93.3%) 59(98.3%) 40(66.7%) 28(46.7%) 44(73.3%)

Nuclear characteristics

• Smudgy Chromatin 0(0%) 0(0%) 1(1.7%) 0(0%) 0(0%)

• Mod crisp

chromatin 5(8.3%) 1(1.7%) 35(58.3%) 42(70%) 14(23.3%)

• Crisp chromatin 55(91.7%) 59(98.3%) 24(40%) 18(30%) 46(76.7%)

Cytoplasmic details

• Unsatisfactory 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Sub-optimal 3(5%) 1(1.7%) 20(33.3%) 35(58.3%) 20(33.3%)

• Optimal 57(95%) 59(98.3%) 40(66.7%) 25(41.7%) 40(66.7%)

Air drying artifacts

• >50% 0(0%) 0(0%) 3(5%) 1(1.7%) 0(0%)

• <50% 21(35%) 21(35%) 13(21.7%) 19(31.7%) 30(50%)

• 0% 39(65%) 39(65%) 44(73.3%) 40(66.7%) 30(50%)

37

FNAC OF 60 THYROID LESIONS YIELDED FOLLOWING RESULTS:

Background was hemorrhagic in 49(81.7%) of cases in MGG and

14(23.3%) of cases in MUFP. Clean background was seen in 46(76.7%) cases of

MUFP cases and in 11(18.3) cases of MGG.

Overall staining was good in 98.3% of Pap, 96.7% of H&E and 60% of

REAP cases. 60% of MUFP cases showed average overall staining.

Cell morphology was well preserved in 98% of Pap, 93.3% of H&E and

73.3% of REAP cases.

Nuclear characteristics with crisp chromatin were seen in 98.3% of Pap,

91.7% of H&E and 76.6% of REAP cases. 70% of MUFP cases showed moderately

crisp chromatin.

Optimal cytoplasmic details were noted in 98.3% of Pap, 95% of H&E and

66.7% of REAP cases. 58.3% of MUFP cases showed sub-optimal cytoplasmic

details.

Air drying artifacts were least with air dried smears like MGG(73.3%) and

air dried rehydrating smears like MUFP(66.7%).

38

TABLE NO 5

BREAST (44 CASES)

H&E

PAP MGG MUFP REAP

Background

• Hemorrhagic 9(20.5%) 13(29.5%) 38(86.4%) 9(20.5%) 21(47.7%)

• Clean 35(79.5%) 31(70.5%) 6(13.6%) 35(79.5%) 23(52.3%)

Overall staining

• Poor 0(0%) 0(0%) 0(0%) 1(2.3%) 0(0%)

• Average 2(4.5%) 2(4.5%) 29(65.9%) 28(63.6%) 9(20.5%)

• Good 42(95.5%) 42(95.5%) 15(34.1%) 15(34.1%) 35(79.5%)

Cell morphology

• Poorly preserved 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Moderately

preserved 1(2.3%) 1(2.3%) 16(36.4%) 24(54.5%) 3(6.8%)

• Well preserved 43(97.7%) 43(97.7%) 28(63.6%) 20(45.5%) 41(93.2%)


• Smudgy Chromatin 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Mod crisp chromatin 3(6.8%) 1(2.3%) 21(47.7%) 33(75%) 5(11.4%)

• Crisp chromatin 41(93.2%) 43(97.7%) 23(52.3%) 11(25%) 39(88.6%)

Cytoplasmic details

• Unsatisfactory 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Sub-optimal 2(4.5%) 1(2.3%) 19(43.2%) 27(61.4%) 7(15.9%)

• Optimal 42(95.5%) 43(97.7%) 25(56.8%) 17(38.6%) 37(84.1%)


• >50% 0(0%) 0(0%) 1(2.3%) 1(2.3%) 0(0%)

• <50% 15(34.1%) 13(29.5%) 15(34.1%) 18(40.9%) 16(36.4%)

• 0% 29(65.9%) 31(70.5%) 28(63.6%) 25(56.8%) 28(63.6%)

39

FNAC OF 44 BREAST LESIONS YIELDED FOLLOWING RESULTS:

Background was clean in 79.5% of MUFP and H&E cases.86.4% of MGG

cases showed hemorrhagic background.

Overall staining was good in 95.5% of Pap and H&E cases. In REAP 79.5%

of cases showed good overall staining. 29 cases(65.9%) of MGG and 28 cases(63.6%)

of MUFP showed average overall staining.

97.7% of Pap and H&E, 93.2% of REAP stained smears showed well

preserved cell morphology. Moderately preserved cell morphology was seen in 54.5%

of MUFP cases.

Crisp nuclear characteristics were noted in 97.7% of Pap, 93.2% of H&E and

88.6% of REAP cases. 75% of MUFP cases showed moderately crisp chromatin.

Cytoplasmic details were optimal in 97.7% of Pap, 95.5% of H&E and 84.1%

of REAP cases. Sub-optimal cytoplasmic details were seen in 61.4% of MUFP cases.

Air drying artifacts are not seen in 70.5% of Pap, 65.9% of H&E, 28% of

MGG and REAP cases. 25% of MUFP cases showed no air dried artifacts.

40

TABLE NO 6

LYMPH NODE (36 CASES)

H&E

PAP MGG MUFP REAP

Background

• Hemorrhagic 12(33.3%) 10(27.8%) 24(66.7%) 5(13.9%) 24(66.7%)

• Clean 24(66.7%) 26(72.2%) 12(33.3%) 31(86.1%) 12(33.3%)

Overall staining

• Poor 0(0%) 0(0%) 0(0%) 3(8.3%) 0(0%)

• Average 3(8.3%) 1(2.8%) 26(72.2%) 19(52.8%) 13(36.1%)

• Good 33(91.7%) 35(97.2%) 10(27.8%) 14(38.9%) 23(63.9%)

Cell morphology


• Moderately

preserved 4(11.1%) 0(0%) 11(30.6%) 10(27.8%) 7(19.4%)

• Well preserved 32(88.9%) 36(100%) 25(69.4%) 26(72.2%) 29(80.6%)



• Mod crisp

chromatin 5(13.9%) 0(0%) 23(63.9%) 24(66.7%) 3(8.3%)

• Crisp chromatin 31(86.1%) 36(100%) 13(36.1%) 12(33.3%) 33(91.7%)

Cytoplasmic details

• Unsatisfactory 0(0%) 0(0%) 0(0%) 1(2.8%) 0(0%)

• Sub-optimal 5(13.9%) 1(2.8%) 13(36.1%) 12(33.3%) 8(22.2%)

• Optimal 31(86.1%) 35(97.2%) 23(63.9%) 23(63.9%) 28(77.8%)


• >50% 0(0%) 0(0%) 1(2.8%) 1(2.8%) 1(2.8%)

• <50% 19(52.8%) 16(44.4%) 12(33.3%) 11(30.6%) 17(47.2%)

• 0% 17(47.2%) 20(55.6%) 23(63.9%) 24(66.7%) 18(50%)

41

FNAC OF 36 LYMPH NODE LESIONS YIELDED FOLLOWING RESULTS:

Background was clean in 86.1% of MUFP cases. Hemorrhagic background

was seen in 66.7% of MGG and REAP cases.

Overall staining was good in 97.2% of Pap, 91.7% of H&E and 63.9% of

REAP cases. Average overall staining was seen in 72.2% of MGG cases.

Well preserved cell morphology was seen in 100% of Pap, 88.9% of H&E,

80.6% of REAP , 72.2% of MUFP and 69.4% of MGG cases.

Crisp nuclear characteristics were noted in 100% of Pap, 91.7% of REAP and

86.1% of H&E cases. Moderately crisp chromatin was noted in 66.7% of MUFP and

63.9% of MGG cases.

Cytoplasmic details were optimal in 97.2% of Pap, 86.1% of H&E, 77.8% of

REAP, 63.9% of MUFP and MGG cases.

Air drying artifacts were not seen in 66.7% of MUFP and 63.9% of MGG

cases. 52.8% of H&E and 47.2% of REAP cases showed <50% air drying artifacts.

42

TABLE NO 7

SALIVARY GLAND (10 CASES)

H&E

PAP MGG MUFP REAP

Background

• Hemorrhagic 7(70%) 7(70%) 9(90%) 2(20%) 6(60%)

• Clean 3(30%) 3(30%) 1(10%) 8(80%) 4(40%)

Overall staining

• Poor 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Average 1(10%) 1(10%) 8(80%) 5(50%) 5(50%)

• Good 9(90%) 9(90%) 2(20%) 5(50%) 5(50%)

Cell morphology


• Moderately

preserved 0(0%) 0(0%) 2(20%) 5(50%) 3(30%)

• Well preserved 10(100%) 10(100%) 8(80%) 5(50%) 7(70%)



• Mod crisp

chromatin 0(0%) 0(0%) 9(90%) 9(90%) 2(20%)

• Crisp chromatin 10(100%) 10(100%) 1(10%) 1(10%) 8(80%)

Cytoplasmic details

• Unsatisfactory 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• Sub-optimal 0(0%) 0(0%) 1(10%) 5(50%) 2(20%)

• Optimal 10(100%) 10(100%) 9(90%) 5(50%) 8(80%)


• >50% 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)

• <50% 4(40%) 4(40%) 4(40%) 4(40%) 7(70%)

• 0% 6(60%) 6(60%) 6(60%) 6(60%) 3(30%)

43

FNAC OF 10 SALIVARY GLAND LESIONS YIELDED FOLLOWING

RESULTS:

Clean background was seen in 80% of MUFP cases. Background was

hemorrhagic in 90% of MGG, 70% of Pap and H&E cases.

Overall staining was good in 90% of Pap and H&E cases. 80% of MGG, 50%

of REAP & MUFP stained smears showed average overall staining.

Cell morphology was well preserved in 100% of Pap, 100% of H&E, 80% of

MGG and 70% of REAP cases.

Nuclear characteristics were crisp in 100% of H&E, 100% of Pap 80% of

REAP stained smears. Moderately crisp chromatin was seen in 90% of MUFP and

MGG cases.

Cytoplasmic details were optimal in 100% of Pap, 100% of H&E, 90% of

MGG and 80% of REAP cases.

Air drying artifacts were not seen in 60% of Pap, H&E, MUFP and MGG

cases each. 70% of REAP cases showed <50% air drying artifacts.

RESULTS OF SPECIFIC STAIN IN DIFFERENT ORGANS:

H&E

score Thyroid

<0.80 3(5%) 4(11.1%)

0.81-1.0 57(95%) 32(88.9%)

Total 60(100%) 36(100%)

Mean ±

SD 0.94±0.07 0.92±0.08

P=0.039* by Fisher Exact test

For H&E Stain, Quality index score difference is statistically significant (p value

<0.05).

Mean Quality index score for H&E stain is maximum for breast followed by thyroid,

salivary gland and lymph node.

GRAPH 3

0

10

20

30

40

50

60

70

80

90

100

Thyroid

Per

cen

tag

e

RESULTS OF SPECIFIC STAIN IN DIFFERENT ORGANS:

TABLE NO 8

H&E STAIN

Lymph

Node Breast

Salivary

Gland Total

4(11.1%) 1(2.3%) 0(0%) 8(5.3%)

32(88.9%) 43(97.7%) 10(100%) 142(94.7%)

36(100%) 44(100%) 10(100%) 150(100%)

0.92±0.08 0.96±0.05 0.93±0.05 0.94±0.07

P=0.039* by Fisher Exact test



gland and lymph node.

GRAPH 3. H&E stain in different organs

Lymph Node

Breast Salivary Gland

0.81-1.0

<0.80

HE Score

44

Total

8(5.3%)

142(94.7%)

150(100%)

0.94±0.07



PAP score Thyroid

<0.80 1(1.7%)

0.81-1.0 59(98.3%) 36(100%)

Total 60(100%) 36(100%)

Mean ±

SD 0.96±0.05 0.95±0.04

P=0.397 by Fisher Exact test

PAP score is positively associated with P value of 0.397.

Mean Quality index score for Pap stain is maximum for breast and thyroid followed

by lymph node and salivary gland.

GRAPH 4

0

10

20

30

40

50

60

70

80

90

100

Thyroid

Per

cen

tag

e

TABLE NO 9

PAP STAIN

Lymph

Node Breast

Salivary

Gland Total

0(0%) 1(2.3%) 0(0%) 2(1.3%)

36(100%) 43(97.7%) 10(100%) 148(98.7%)

36(100%) 44(100%) 10(100%) 150(100%)

0.95±0.04 0.96±0.06 0.93±0.05 0.95±0.05


associated with P value of 0.397.


node and salivary gland.

GRAPH 4. PAP stain in different organs

Lymph Node


0.81-1.0

<0.80

PAP Score

45

Total

2(1.3%)

148(98.7%)

150(100%)

0.95±0.05


MGG

score Thyroid

<0.80 32(53.3%) 14(38.9%)

0.81-1.0 28(46.7%) 22(61.1%)

Total 60(100%) 36(100%)

Mean ±

SD 0.81±0.11 0.81±0.09


Mean Quality index score for MGG stain is maximum for breast,thyroid and lymph

node followed by salivary gland.

GRAPH 5

0

10

20

30

40

50

60

70

80

90

100

Thyroid

Per

cen

tag

e

TABLE NO 10

MGG STAIN

Lymph

Node Breast

Salivary

Gland Total

14(38.9%) 18(40.9%) 4(40%) 68(45.3%)

22(61.1%) 26(59.1%) 6(60%) 82(54.7%)

36(100%) 44(100%) 10(100%) 150(100%)

0.81±0.09 0.81±0.09 0.80±0.08 0.80±0.09



node followed by salivary gland.

GRAPH 5. MGG stain in different organs

Lymph Node


0.81-1.0

<0.80

MGG Score

46

Total

68(45.3%)

82(54.7%)

150(100%)

0.80±0.09


MUFP

score Thyroid

<0.80 32(53.3%) 13(36.1%)

0.81-1.0 28(46.7%) 23(63.9%)

Total 60(100%) 36(100%)

Mean ±

SD 0.82±0.12 0.85±0.12


Mean Quality index score for MUFP stain is maximum for lymph node followed by

thyroid, salivary gland and breast.

Nuclear stain used in MUFP is Gill’s hematoxylin. For 10 cases of FNAC smears,

Harri’s hematoxylin was used in comparision with G

similar results were found.

GRAPH 6. MUFP stain in different organs:

0

10

20

30

40

50

60

70

80

90

100

Thyroid

Per

cen

tag

e

TABLE NO 11

MUFP STAIN

Lymph

Node Breast

Salivary

Gland Total

13(36.1%) 26(59.1%) 4(40%) 75(50%)

23(63.9%) 18(40.9%) 6(60%) 75(50%)

36(100%) 44(100%) 10(100%) 150(100%)

0.85±0.12 0.80±0.12 0.82±0.09 0.82±0.12

Exact test


salivary gland and breast.


Harri’s hematoxylin was used in comparision with Gill’s hematoxylin in MUFP and

GRAPH 6. MUFP stain in different organs:

Lymph Node


0.81-1.0

<0.80

MUFP Score

47

Total

75(50%)

75(50%)

150(100%)

0.82±0.12



ill’s hematoxylin in MUFP and

REAP

score Thyroid

<0.80 17(28.3%) 7(19.4%)

0.81-1.0 43(71.7%) 29(80.6%)

Total 60(100%) 36(100%)

Mean ±

SD 0.85±0.10 0.88±0.09


For REAP stain, score difference is statistically significant and more

lymph node and breast with P value of 0.007.

Mean Quality index score for REAP stain is maximum for breast followed by lymph

node, thyroid and salivary gland.

GRAPH 7. REAP stain in different organs

0

10

20

30

40

50

60

70

80

90

100

Thyroid

Per

cen

tag

e

TABLE NO 12

REAP STAIN

Lymph

Node Breast

Salivary

Gland Total

7(19.4%) 1(2.3%) 4(40%) 29(19.3%)

29(80.6%) 43(97.7%) 6(60%) 121(80.7%)

36(100%) 44(100%) 10(100%) 150(100%)

0.88±0.09 0.92±0.06 0.85±0.13 0.88±0.09


For REAP stain, score difference is statistically significant and more associated with

lymph node and breast with P value of 0.007.


node, thyroid and salivary gland.

GRAPH 7. REAP stain in different organs

Lymph Node


0.81-1.0

<0.80

REAP Score

48

Total

29(19.3%)

121(80.7%)

150(100%)

0.88±0.09

associated with


Correlation of Background

Based on total number of units , 600 samples are considered for significance

GRAPH 8. Correlation of Background in H & E, PAP,

0

10

20

30

40

50

60

70

80

90

100

H &E PAP

Per

cen

tag

e

BACKGROUND H&E

HEMORRHAGIC 51(34%)

CLEAN 99(66%)

TOTAL 150(100%)

Test Applied Value

Chi Square Test 129

TABLE NO 13

Correlation of Background in H & E, PAP, MGG, MUFP and REAP


Correlation of Background in H & E, PAP, MGG, MUFP and REAP

MGG MUFP REAP

Clean

Hemorrhagic

Background

PAP MGG MUFP REAP

48(32%) 120(80%) 30(20%) 95(63.3)

102(68%) 30(20%) 120(80%) 55(36.7)

150(100%) 150(100%) 150(100%) 150(100%) 150(100%)

Value P value Difference is

<0.001 Significant

49

in H & E, PAP, MGG, MUFP and REAP


MGG, MUFP and REAP

Hemorrhagic

REAP

95(63.3)

55(36.7)

150(100%)

Correlation of Overall staining in H & E, PAP, MGG, MUFP and REAP


GRAPH 9. Correlation of overall

0

10

20

30

40

50

60

70

80

90

100

H &E PAP

Per

cen

tag

eOVERALL

STAINING

H&E

POOR 0(0%)

AVERAGE 8(5.3%)

GOOD 142(94.7%)

TOTAL 150(100%)

Test Applied Value

Chi Square Test 250

TABLE NO 14

of Overall staining in H & E, PAP, MGG, MUFP and REAP


Correlation of overall staining in H & E, PAP, MGG,

MUFP and REAP

PAP MGG MUFP REAP

GOOD

AVERAGE

POOR

Overall staining

PAP MGG MUFP REAP

0(0%) 2(1.3%) 4(2.6%) 0(0%)

5(3.3%) 102(68%) 88(58.7%) 51(34%)

145(96.7%) 46(30.7%) 58(38.7%) 99(66%)

150(100%) 150(100%) 150(100%) 150(100%)


250 <0.001 Significant

50

of Overall staining in H & E, PAP, MGG, MUFP and REAP


H & E, PAP, MGG,

REAP

0(0%)

51(34%)

99(66%)

150(100%)

Correlation of Cell Morphology in H & E, PAP, MGG, MUFP and REAP


GRAPH 10. Correlation of cell morphology in H & E, PAP, MGG, MUFP and

0

10

20

30

40

50

60

70

80

90

100

H &E PAP

Per

cen

tag

e

CELL

MORPHOLOGY

H&E

POORLY

PRESERVED

0(0%)

MODERATELY

PRESERVED

9(6%)

WELL

PRESERVED

141(94%)

TOTAL 150(100%)

Test Applied Value

Chi Square Test 127

TABLE NO 15



Correlation of cell morphology in H & E, PAP, MGG, MUFP and

REAP

PAP MGG MUFP REAP

Well preserved

Moderately preserved

Poorly preserved

Cell

PAP MGG MUFP REAP

0(0%) 1(0.6%) 1(0.6%) 0(0%)

2(1.3%) 48(32%) 70(46.7%) 29(19.3%)

148(98.7%) 101(67.4%) 79(52.7%) 121(80.7%)

150(100%) 150(100%) 150(100%) 150(100%) 150(100%)



51


Correlation of cell morphology in H & E, PAP, MGG, MUFP and

REAP

0(0%)

29(19.3%)

121(80.7%)

150(100%)

Correlation of Nuclear characteristics in H & E, PAP, MGG, MUFP and REAP

Based on total number of units , 600 samples are considered for statistical significance

GRAPH 11. Correlation of nuclear characteristics in H & E, PAP, MGG,

0

10

20

30

40

50

60

70

80

90

100

H &E PAP

Per

cen

tag

e

NUCLEAR

CHARACTERISTICS

H&E

SMUDGY

CHROMATIN

0(0%)

MODERATELY

CRISP CHROMATIN

13(8.6%)

CRISP CHROMATIN 137(91.4%)

TOTAL 150(100%)

Test Applied Value

Chi Square Test 251

TABLE NO 16



Correlation of nuclear characteristics in H & E, PAP, MGG,

and REAP

PAP MGG MUFP REAP

crisp

Moderately Crisp

Smudgy


PAP MGG MUFP

0(0%) 1(0.6%) 0(0%)

13(8.6%) 2(1.3%) 88(58.7%) 108(72%)

137(91.4%) 148(98.7%) 61(40.7%) 42(28%)

150(100%) 150(100%) 150(100%) 150(100%)



52



Correlation of nuclear characteristics in H & E, PAP, MGG, MUFP

REAP

0(0%)

24(16%)

126(84%)

150(100%)

53

TABLE NO 17

Correlation of cytoplasmic details in H & E, PAP, MGG, MUFP and REAP


GRAPH 12. Correlation of cytoplasmic details in H & E, PAP, MGG, MUFP and

REAP

CYTOPLASMIC

DETAILS

H&E PAP MGG MUFP REAP

UNSATISFACTORY 0(0%) 0(0%) 0(0%) 1(0.6%) 0(0%)

SUB-OPTIMAL 10(6.7%) 3(2%) 53(35.3%) 79(52.7%) 37(24.7%)

OPTIMAL 140(93.3%) 147(98%) 97(64.7%) 70(46.7%) 113(75.3%)

TOTAL 150(100%) 150(100%) 150(100%) 150(100%) 150(100%)

Test Applied Value P value Difference is

Chi Square Test 144 <0.001 Significant

Correlation of air drying artifacts in H & E, PAP, MGG, MUFP and REAP


GRAPH 13. Correlation of air drying artifacts in H & E, PAP, MGG, MUFP and

0

10

20

30

40

50

60

70

80

90

100

H &E PAP

Per

cen

tag

e

AIR DRYING

ARTIFACTS

H&E

• >50% 0(0%)

• <50% 59(39.3%)

• 0% 91(60.7%)

TOTAL 150(100%)

Test Applied Value

Chi Square Test 1.47

TABLE NO18



Correlation of air drying artifacts in H & E, PAP, MGG, MUFP and

REAP

MGG MUFP REAP

>50% <50% 0%

AIR DRYING ARTIFACTS

PAP MGG MUFP REAP

0(0%) 5(3.3%) 3(2%) 1(0.6%)

54(36%) 44(29.3%) 52(34.7%) 70(46.7%)

96(64%) 101(67.4%) 95(63.3%) 79(52.7%)

150(100%) 150(100%) 150(100%) 150(100%)


1.47 0.690 Not Significant

54



Correlation of air drying artifacts in H & E, PAP, MGG, MUFP and

0%

REAP

1(0.6%)

70(46.7%)

79(52.7%)

150(100%)

55

Statistical Methods: Descriptive and inferential statistical analysis has been carried

out in the present study. Results on continuous measurements are presented on Mean

± SD (Min-Max) and results on categorical measurements are presented in Number

(%). Significance is assessed at 5 % level of significance.

Chi-square/ Fisher Exact test has been used to find the significance of study

parameters on categorical scale between two or more groups.

Significant figures:

+ Suggestive significance (P value: 0.05<P<0.10)

* Moderately significant ( P value:0.01<P ≤ 0.05)

** Strongly significant (P value : P≤0.01)

Statistical software: The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1,

MedCalc 9.0.1 ,Systat 12.0 and R environment ver.2.11.1 were used for the analysis

of the data and Microsoft word and Excel have been used to generate graphs, tables

etc.

56

FIGURE 1. HASHIMOTO’S THYROIDITIS, PAPANICOLAOU st ain, 100X

FIGURE 2. HASHIMOTO’S THYROIDITIS,H&E stain, 100 X.

57

FIGURE 3. HASHIMOTO’S THYROIDITIS, Hurthle cell cha nge, H&E stain, 400x

FIGURE 4. HASHIMOTO’S THYROIDITIS,MGG STAIN, 400X

58

FIGURE 5. HASHIMOTO’S THYROIDITIS, REAP stain, 400 X

FIGURE 6. HASHIMOTO’S THYROIDITIS, MUFP stain, 400 x

59

FIGURE 7. FIBROADENOMA OF BREAST, Pap stain,400x

FIGURE 8. FIBROADENOMA OF BREAST, H&E stain, 400x

60

FIGURE 9. FIBROADENOMA OF BREAST, MGG stain, 100x and 400x

FIGURE 10(a) and (b) FIBROADENOMA OF BREAST, REAP, 100x and 400X

FIGURE 11(a) and (b) FIBROADENOMA OF BREAST, MUFP stain, 100X

FIGURE 12. CARCINOMA OF BREAST, Pap stain, 100x and 400x

FIBROADENOMA OF BREAST, MUFP stain, 100X and 400x


61

FIBROADENOMA OF BREAST, MUFP stain, 100X


62

FIGURE 13(a) and (b) CARCINOMA OF BREAST, H&E,100x and 400x

FIGURE 14(a) and (b) CARCINOMA OF BREAST, MGG,100x and 400x

63

FIGURE 15.CARCINOMA OF BREAST, REAP stain, 400x

FIGURE 16.CARCINOMA OF BREAST, MUFP stain, 400x

64

FIGURE 17.TUBERCULOUS LYMPHADENITIS, Pap stain,400x

FIGURE 18.TUBERCULOUS LYMPHADENITIS, H&E stain,400 x

65

FIGURE 19.TUBERCULOUS LYMPHADENITIS, MGG stain,400 x

FIGURE 20.TUBERCULOUS LYMPHADENITIS, REAP stain,40 0x

66

FIGURE 21.TUBERCULOUS LYMPHADENITIS, MUFP stain, 4 00x

FIGURE 22. SQUAMOUS CELL CARCINOMA SECONDARIES IN L YMPH NODE, Pap stain, 400x

67

FIGURE 23. SQUAMOUS CELL CARCINOMA SECONDARIES IN L YMPH NODE, H&E stain, 400x

FIGURE 24. SQUAMOUS CELL CARCINOMA SECONDARIES IN L YMPH NODE, MGG stain, 400x

68

FIGURE 25. SQUAMOUS CELL CARCINOMA SECONDARIES IN L YMPH NODE, REAP stain, 400x

FIGURE 26. SQUAMOUS CELL CARCINOMA SECONDARIES IN L YMPH NODE, MUFP stain, 400x

69

FIGURE 27. PLEOMORPHIC ADENOMAOF PAROTID, Pap stain ,400x

FIGURE 28. PLEOMORPHIC ADENOMAOF PAROTID, H&E stain ,400x

70

FIGURE 29(a) and (b) PLEOMORPHIC ADENOMAOF PAROTID, MGG stain, 100x and 400x

FIGURE 30(a) and (b) PLEOMORPHIC ADENOMAOF PAROTID , REAP stain, 100x and 400x

71

FIGURE 31(a) PLEOMORPHIC ADENOMAOF PAROTID, MUFP s tain, 100x

FIGURE 31(b) PLEOMORPHIC ADENOMAOF PAROTID, MUFP s tain, 400x

72

DISCUSSION

Fine needle aspiration cytology (FNAC) is one of the less expensive(economical),

fastest and easiest tools available for early detection and diagnosis of various lesions. Since its

inception, PAP stain remains the traditional and preferred stain, not only for the gynecological

cytology, but also for the lesions of other organs.

The different stains used for air dried smears, such as May-Grunwald-Giemsa,

Jenner-Giemsa and Diff-Quick fail to offer the transparency for the study of subtle nuclear

features as seen by the PAP stain.

The traditional pap stain involves wet fixation and subsequent staining, together

requiring at least 30 minutes. To cut down the time, the rapid pap stains were developed by

kline, Tao and sato with respective staining time of 4 minutes, 5 minutes and 90 seconds.

However, the quality of rapid papanicolaou staining is usually not satisfactory, as the cell

morphology is not well seen.

To overcome these problems, Yang and Alvarez developed ultra-Fast pap (UFP) stain

which is a hybrid of papanicolaou and Romanowsky stains. The staining time of UFP is 90

seconds. Kamal et al from India further modified the UFP stain (Modified ultra-Fast pap

stain) to overcome the problem of shortage of Richard-Allan hematoxylin and Richard-Allan

cytostain in Indian set-up. This method has a short staining time of 130 seconds, and the

cytomorphology can be well appreciated.

In the present study cytomorphology of rehydrated air dried smears followed by

modified ultrafast papanicolaou staining was compared with methanol fixed smear stained by

REAP stain and ethyl alcohol fixed smears stained by conventional pap stain, H & E stain and

air-dried smears stained by MGG.

73

The quality of all 5 stains was evaluated on 6 parameters such as background, overall

staining, cell morphology, nuclear characteristics, cytoplasmic features and air-drying

artifacts.

The quality index of different organs i.e., thyroid, Breast, lymph node and Salivary

gland in all five stains were calculated.

Shinde et al. calculated quality index of four sites, lymphnode, breast, thyroid and

salivary gland for ultrafast pap stain as follows.29 The Quality indices in this study are

comparable to our study.

The number of cases studied by us is considerably more than Shinde’s study.

Table 19. Quality index in different organs in Shinde’s study and present study

for MUFP stain:

ORGAN SHINDE’S STUDY PRESENT STUDY

NO. OF CASES QUALITY INDEX NO. OF CASES QUALITY INDEX

THYROID 8 0.98 60 0.82

BREAST 16 0.92 44 0.80

LYMPH NODE 15 0.98 36 0.85

SALIVARY

GLAND

2 0.95 10 0.82

74

Priyanka Choudhary et al. Calculated quality index scores of MUFP in four organs and

compared with rapid papanicolaou stain.33

Table 20. Quality index in different organs in Priyanka’s study

ORGAN No. of CASES QUALITY INDEX OF

MUFP

Thyroid 25 1

Breast 23 0.97

Lymphnode 43 0.98

Soft tissue and others 9 1

Alkhair Abd Almahmoud Idris48 compared the efficacy of three stains, PAP, H & E &

MGG in FNAC of breast lumps. In this study pap stain showed the maximum quality index

score followed by H & E and MGG which is comparable with our study.

Table 21.Quality Index scores of Breast in Almahmoud Idris study and present study

PAP H & E MGG

Almahmoud Idris

study

0.87 0.81 0.77

Present study 0.96 0.94 0.81

75

Gupta et al.38 did a study on modified papanicolaou staining protocol with minimum

alcohol use. In this study REAP stain showed crisp nuclear chromatin in 73.3% of cases. This

is comparable with our study and its is 84%.

Table 22. Nuclear features in Gupta’s study and present study with REAP

Crisp Chromatin Hazy chromatin

Gupta et al study 73.3% 26.7%

Present study 84% 16%

BACKGROUND :

Heavy blood staining is a commonly encountered problem in conventionally fixed

and stained preparations, which can be overcome by Rehydration technique. In the present

study as shown in table 18, air dried smears rehydrated with normal saline as in MUFP show

minimum hemorrhage of 20% as compared to wet fixed smears PAP (32%), H & E (34%%),

REAP (63.3%) and air dried smears MGG (80%). 80% of smears in MUFP show clean

background. The P value <0.001 calculated by applying chi-suqare test proved the difference

to be significant. .

These findings are comparable to findings of other studies. Shinde et al. showed that

95% of the smears in MUFP showed clean background.29 Choudhary P. et al. found that

MUFP stained smears has clean, RBC free background and thus helps in better interpretation

in vascular organs like thyroid.33 Maruta et al. reported that MUFP stain lyses red blood cells

in the background, make the smear thinner and clearer. This makes the slide better for cyto

morphologic observation.22

The Rehydration solution, normal saline discovered by Chang and Kung39 restores

the transparency of air-dried cells and thus their nuclear details reappear. In addition it

76

hemolyses the RBC’s in the smear and unmasks tumor cells.23 Cells were larger because of

air-drying, nucleoli were distinctly seen and stain red.25

The present study confirms that air-dried smears rehydrated with normal saline

provide clean background as compared to air-dried smears and wet fixed smears.

OVERALL STAINING:

As per table 14, the overall staining is good and maximum for Pap stain (96.7%)

followed by H&E (94.7%), REAP (66%), MUFP (38.7%) and MGG (30.7%). The value of P

is <0.001, thus making the difference significant.

This is comparable to study by Almahmoud Idris. Intheir study overall staining

score is maximum for Pap followed by H&E and MGG.48

CELL MORPHOLOGY:

As per table 15, Cell morphology is well preserved and maximum for Pap stain

(98.4%) followed by H&E (94%), REAP (80.7%), MGG (67.4%) and MUFP (52.7%).

Statistically the difference is significant as P value is <0.001.

This is comparable to study by Almahmoud Idris. In this study cell morphology

score is maximum for Pap followed by H&E and MGG.48

NUCLEAR CHARACTERISTICS:

The nuclei were assessed for crispness of chromatin. Nuclei were graded as smudgy

chromatin, moderately crisp and crisp chromatin.

As per table 16, value of P is <0.001 thus making difference statistically significant.

The nuclear characteristics were crisp in 98.7% of Pap stained smears followed by H&E

(91.4%), REAP (84%), MGG (40.7%) and MUFP (28%).

77

This is comparable to study by Almahmoud Idris. In this study crisp nuclear features

are seen in Pap stain followed by H&E and MGG.48

Gupta et al. showed that crisp nuclear features are seen in 73.3% of REAP cases

studied.38 Biswas et al. reported 100 smears out of 110 Pap smears stained with REAP are

optimal.36 Dighe et al. showed that 192 Pap smears out of 200 stained with REAP are

optimal.37

Yang et al. reported that by highlighting the Orphan-Annie-eyed clear nuclei,

Ultrafast Pap stain easily distinguishes follicular variant of papillary thyroid carcinoma from

follicular neoplasms.28

CYTOPLASMIC DETAILS:

The cytoplasmic features are graded as unsatisfactory, suboptimal and optimal. As

per table 17 cytoplasmic features are better and optimal in 98% of Pap stained smears

followed by H&E (93.3%), REAP (75.3%), MGG (64.7%) and MUFP (46.7%). Value of P is

<0.001 thus making difference statistically significant

Dighe et al. reported that 181 Pap smears out of 200 stained with REAP showed

optimal cytoplasmic features.37 Biswas et al.reported 100 smears out of 110 Pap smears

stained with REAP showed optimal cytoplasmic stain.36

The cytoplasm is stained in different shades of green, pink, red to orange in

Papanicolaou stain and in REAP stain. Cytoplasmic features are more informative in Pap

stain than in routine H&E.

As Orange G was omitted from staining solution in Modified ultrafast stain, it is to be

used in tissues where chances of cytoplasmic keratinization is negligible.

78

AIR-DRYING ARTIFACTS:

As per table 18, Air- drying artifacts are less in air dried smears. 67.4% of MGG

smears show no air drying artifacts followed by Pap (64%), MUFP (63.3%), H&E (60.7%)

and REAP (52.7%). Statistically the difference is not significant as P value is 0.690.

Kamal et al. showed that the problem of wet fixation, air drying artifacts can be

eliminated by rehydration of air dried smears as in MUFP and the physical hurry for

immeadiate wet fixation was no longer essential.25

Thus our study proved that air drying technique and rehydration of air dried smears

was associated with less air drying artifacts as compared to wet fixation.

The amount of air drying artifacts also depends upon skill of person who makes the

smear. Thus, rehydration technique allows less skilled person to make smears leisurely

without any apprehension about the appearance of these artifacts.

Our study compares the modifications of Papanicolaou stain like Modified Ultrafast

Papanicolaou Stain (MUFP) and Rapid Economic Acetic acid Papanicolaou stain with routine

stains used in cytology like conventional Papanicolaou stain, H&E stain and MGG stain.

MUFP is rapid and has the advantage of clean background and less air drying artifacts. REAP

stain require limited amount of alcohol, it is rapid and economical.

79

SUMMARY

• This prospective study was carried over a period of 21 months, which

included 150 cases ( Fine Needle Aspiration Cytology from organs like

Thyroid, Breast, Lymph node and Salivary gland ).

• Five different Stains, Papanicolaou stain, Hematoxylin & Eosin stain, May-

Grunwald Giemsa, Modified Ultrafast Papanicolaou stain, Rapid Economic

Acetic acid Papanicolaou stain were compared with each other.

• Smears were compared in six different parameters and significance of

difference was calculated by applying Chi-square/ Fisher Exact test to find the

significance of study parameters on categorical scale between two or more

groups.

Statistical Results with specific organ with inter-stain study

> Means better than

• Of all the cytological stains we did for thyroid, lymph node and salivary gland

lesions, highest quality index score was seen in Pap stain followed by H&E,

REAP, MUFP and MGG.

Thyroid cases - PAP > H&E > REAP >MUFP > MGG

Breast cases - PAP > H&E > REAP >MGG > MUFP

Lymph Node - PAP > H&E > REAP >MUFP > MGG

Salivary Gland - PAP / H&E > REAP >MUFP > MGG

80

• In breast lesions Pap stain got the maximum quality index score followed by

H&E, REAP, MGG and MUFP.

Statistical Results with specific stain with inter-organ study

H&E stain - Breast > Thyroid > Salivary Gland > Lymph Node

PAP stain - Thyroid/ Breast > Lymph Node > Salivary Gland

MGG stain - Thyroid/ Lymph Node/ Breast > Salivary Gland

MUFP stain - Lymph Node > Thyroid / Salivary Gland > Breast

REAP stain - Breast > Lymph Node > Thyroid / Salivary Gland

> means better than

• Pap stain was best for thyroid and breast lesions followed by lymph node and

salivary gland.

• H&E stain was found to be best for all breast lesions followed by thyroid,

salivary gland and lymph node.

• MGG stain was found to be good for thyroid, lymph node, and breast lesions.

It is average for salivary gland lesions.

• MUFP stain was found to be best for all inflammatory lesions and also for

lymph node lesions.

• REAP stain gave good results for breast lesions followed by lymph node,

thyroid, and salivary gland lesions.

81

CONCLUSION

� Papanicolaou stain is excellent in studying FNAC material of all four organs

,namely thyroid, breast, lymphnode and salivary gland lesions . It got

maximum score in all five parameters compared except for air drying artifacts.

� H&E as a routine and easily available stain has good score, but still is not as

good as conventional Papanicolaou stain.

� MGG stained smears showed less air drying artifacts when compared to wet

fixed smears and air dried smears rehydrated with normal saline as in MUFP.

� REAP stain is as good as Papanicolaou stain but it has the disadvantage of

hemorrhagic background and more air drying artifacts. The nuclear

characteristics are crisp in REAP which is comparable with that of Pap stain.

� MUFP stained smears showed clean background and less air drying artifacts.

The alcoholic formalin used in MUFP takes lesser staining time for fixation

and makes nucleoli to appear red and prominent.

� Compared to conventional Papanicolaou stain, REAP is economical as 1%

acetic acid which is easily available is used instead of alcohol. Staining time is

also lesser in REAP.

� Air drying artifacts are less in air-dried smears like MGG and air-dried,

rehydrated smears as in MUFP compared to wet fixed smears.

� REAP gives excellent nuclear morphology especially in breast lesions.

� MUFP is very good stain for inflammatory and lymph node lesions.

� REAP and MUFP stains can be used as routine cytological stains.

82

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88

CASE PROFORMA

Name : Age :

Sex : IP/OP :

Unit: Cyt no:

Clinical presentation:

Past history:

Family history:

Personal history:

General physical examination:

Pallor Lymphadenopathy

Icterus Cyanosis

Clubbing Oedema

Pulse rate:

Blood pressure:

Respiratory rate:

Systemic examination:

RS CVS Per abdomen Investigations:

89

Local examination: Clinical Diagnosis: Nature of Aspirate:

Microscopy :

Background Overall staining

Cell morphology


Cytoplasmic details


H&E

PAP

MGG

MUFP

REAP

1.Background 2.Overall Staining

Hemorrhagic-Score 1 Poor -Score1

Clean -Score 2 Average -Score 2

Good -Score 3

90

3.Cell Morphology 5.Cytoplasmic Details

Poorly Preserved -Score 1 Unsatisfactory-Score 1

Moderately Preserved-Score 2 Suboptimal -Score 2

Well Preserved -Score 3 Optimal -Score 3

4.Nuclear Characteristics 6.Air Drying Artifacts

Smudgy Chromatin -Score 1 >50%-Score 1

Moderately Crisp Chromatin-Score 2 <50%-Score 2

Crisp Chromatin -Score 3 0% -Score 3

91

KEY TO MASTER CHART

SCORING SYSTEM USED IN ASSESSMENT OF STAINING:

PARAMETER SCORE=1 SCORE=2 SCORE=3

BACKGROUND HEMORRHAGE CLEAN

OVERALL STAINING POOR AVERAGE GOOD

CELL MORPHOLOGY POORLY PRESERVED

MODERATELY PRESERVED

WELL PRESERVED

NUCLEAR CHARACTERISTICS SMUDGY CHROMATIN

MODERATELY CRISP CHROMATIN

CRISP CHROMATIN

CYTOPLASMIC DETAILS UNSATISFACTORY SUBOPTIMAL OPTIMAL

AIR DRYING ARTIFACTS >50% <50% 0%

• Total score is sum of the scores of all 6 parameters. Maximum score for a

single case is 17.

• The “Quality Index” was obtained by calculating the ratio of total score

obtained to the maximum score possible.

Quality Index= actual score obtained /maximum score possible

HEMATOXYLIN AND EOSIN STAIN PAPANICOLAOU STAIN MAY GRUNWALD GIEMSA MODIFIED ULTRAFAST PAPANICOLAOU STAIN

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1 1368/12 27 Female Thyroid Nodular Goiter 1 2 2 2 3 3 0.7 1 3 3 3 3 3 0.94 1 1 2 2 2 2 0.58 2



4 1375/12 40 Male Thyroid Thyroglossal cyst 2 3 3 3 3 3 1 2 2 2 2 2 3 0.76 2 2 1 2 2 1 0.58 2

5 1464/12 55 Female Thyroid Colloid Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 2

6 2145/12 36 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 3 3 2 0.76 2

7 2186/12 27 Female Thyroid Colloid Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 3 3 0.76 2


9 2217/12 22 Female Thyroid Hashimoto's Thyroiditis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 3 3 0.76 2

10 2262/12 40 Female Thyroid Hashimoto's Thyroiditis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 3 2 3 2 0.88 2



13 2420/12 68 Female Thyroid Nodular Goiter 1 2 2 2 2 3 0.7 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2

14 2451/12 20 Female Thyroid Lymphocytic Thyroiditis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 2 0.65 1

15 2453/12 37 Female Thyroid Papillary Carcinoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 1 2 1 0.52 2




19 2511/12 28 Female Thyroid Lymphocytic Thyroiditis 1 3 2 2 2 3 0.76 2 3 3 3 3 3 1 1 3 3 3 3 3 0.88 2

20 2512/12 40 Male Thyroid Nodular Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 2 2 2 3 0.65 1





25 2594/12 40 Female Thyroid Hashimoto's Thyroiditis 1 3 3 3 3 3 0.94 2 3 3 3 3 3 1 1 2 3 2 3 3 0.76 2







32 2693/12 32 Female Thyroid Lymphocytic Thyroiditis 1 3 3 3 3 3 0.94 2 3 3 3 3 3 1 1 2 2 2 2 3 0.65 1



35 99/13 80 Male Thyroid Nodular Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 2 3 3 3 3 0.94 2

36 103/13 21 Female Thyroid Hashimoto's Thyroiditis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 3 2 3 3 0.82 2


38 186/13 45 Female Thyroid Nodular Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 3 3 3 3 3 1 2


40 270/13 42 Female Thyroid Lymphocytic Thyroiditis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 2 2 3 3 0.7 2


42 293/13 41 Male Thyroid Colloid Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.82 1









51 1070/13 70 Female Thyroid ?Nodular goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 2 2 2 3 0.76 2


53 1120/13 40 Female Thyroid Nodular goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 3 2 3 3 3 0.94 2





58 1418/13 36 Female Thyroid Nodular Goitre 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 3 0.76 1



61 1639/12 38 Female Breast Carcinoma Of Breast 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 2 2 2 2 0.65 2

62 1640/12 44 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 1 3 3 3 3 3 0.94 1 2 2 2 2 1 0.58 2

63 1931/12 29 Female Breast Fibroadenoma 1 3 3 3 3 2 0.88 1 3 3 3 3 3 0.88 1 3 3 3 3 3 0.94 2

64 1939/12 44 Female Breast Fibrocystic Disease 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 3 2 2 3 0.82 2

65 2113/12 35 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2


67 2158/12 42 Female Breast Fibroadenoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 3 3 3 0.76 2

68 2182/12 55 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 3 3 3 0.76 2








76 2354/12 30 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 1 2 2 2 2 3 0.7 1 2 2 2 2 3 0.65 2

77 2372/12 30 Female Breast Fibrocystic Disease 1 2 3 2 2 3 0.76 2 3 3 3 3 3 1 1 2 2 2 2 3 0.65 2

78 2389/12 36 Female Breast Fibroadenoma 2 3 3 2 3 3 0.94 2 3 3 3 3 3 1 1 2 2 2 2 3 0.65 2


80 2446/12 53 Male Breast Acute Suppurative Process 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 2

81 2448/12 21 Female Breast Lactational Nodule 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 2 0.65 2



84 2537/12 47 Female Breast Acute Suppurative Process 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.88 1

85 2540/12 22 Female Breast Breast Abscess 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2

86 2553/12 44 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 1 3 3 3 3 3 0.94 1 2 3 3 3 2 0.82 1


88 2562/12 50 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 1 2 3 3 3 3 0.88 1 2 3 3 2 3 0.82 1

89 2609/12 30 Female Breast Granulomatous Mastitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 2







96 232/13 40 Female Breast Fibrocystic Disease 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.82 2

97 344/13 50 Female Breast Tuberculous Abscess 2 3 3 3 3 2 0.94 1 3 3 3 3 2 0.88 1 3 3 3 3 3 0.94 2




101 1143/13 43 Female Breast Benign Cystic Lesion 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 3 3 3 0.94 2



104 1496/13 35 Female Breast Fibroadenoma 2 2 2 2 2 2 1 1 3 3 3 3 3 0.94 1 3 3 2 3 3 0.82 2

105 1373/12 72 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 2 3 3 2 3 0.88 2

106 1482/12 39 Male Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 1 0.58 2

107 1633/12 40 Male Cervical Lymph NodeTuberculous Lymphadenitis 1 2 2 2 2 3 0.7 1 3 3 3 3 3 0.94 1 2 2 2 2 3 0.65 1

108 2012/12 66 Female Cervical Lymph NodeMalignancy-Squamous Cell Carcinoma Secondaries2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 3 0.65 2

109 2194/12 70 Male Cervical Lymph NodeMalignancy-Squamous Cell Carcinoma Secondaries2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 3 2 0.7 1

110 2203/12 52 Male Cervical Lymph NodeLymphoma 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 3 3 2 3 2 0.82 1

111 2225/12 30 Female Cervical Lymph NodeGranulomatous Lymphadenitis 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 2 0.76 1

112 2248/12 50 Female Cervical Lymph NodeReactive Lymphadenitis 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 2 0.76 2

113 2260/12 39 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 3 3 3 0.94 2


115 2305/12 18 Male Inguinal Lymph NodeAcute Suppurative Process 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 3 3 3 3 0.94 2


117 2351/12 35 Male Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 3 1 1 3 3 3 3 3 0.94 2 2 2 2 2 3 0.7 2

118 2368/12 34 Male Cervical Lymph NodeReactive Lymphadenitis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 3 2 2 2 0.76 2


120 2386/12 50 Male Cervical Lymph NodeMalignancy 2 3 3 2 2 3 0.88 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 2

121 2392/12 40 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 2 2 2 2 2 0.7 2 3 3 3 3 3 1 2 2 3 3 2 3 0.82 2

122 2393/12 45 Male Cervical Lymph NodeMalignancy 1 3 2 2 2 3 0.76 2 3 3 3 3 3 1 1 2 2 2 3 2 0.7 2

123 2701/12 16 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 2 2 2 2 0.7 1

124 2706/12 70 Male Inguinal Lymph NodeLymphoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 2

125 2734/12 35 Female Cervical Lymph NodeReactive Lymphadenitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2

126 2779/12 60 Male Cervical Lymph NodeMalignancy-Poorly Differentiated Carcinoma2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 3 3 3 0.88 2

127 93/13 20 Male Cervical Lymph NodeAcute Suppurative Process 2 3 3 3 3 2 0.94 2 3 3 3 2 2 0.88 2 2 3 3 3 3 0.88 2


129 126/13 60 Male Cervical Lymph NodeMalignancy-Squamous Cell Carcinoma Secondaries2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 2 3 0.76 2

130 187/13 47 Male Cervical Lymph NodeReactive Lymphadenitis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 3 2 3 3 0.82 2



133 254/13 20 Female Cervical Lymph NodeReactive Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.82 2

134 267/13 50 Male Cervical Lymph NodeMalignancy-Squamous Cell Carcinoma Secondaries2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 2 2 3 3 0.82 2

135 316/13 50 Male Axillary Lymph Node Reactive Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 2 3 3 0.88 2

136 1084/13 51 Male Cervical Lymph NodeMalignancy-Poorly Differentiated Carcinoma1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 2 2 3 2 0.76 2

137 1095/13 52 Female Cervical Lymph NodeMalignancy 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 2 3 3 0.88 2

138 1351/13 42 Male Cervical Lymph NodeAcute Suppurative Process 1 3 3 3 3 2 0.88 2 3 3 3 3 3 1 2 3 3 3 3 2 0.94 2

139 1383/13 45 Female Pre-auricular Lymph NodeLymphoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 2 3 3 0.82 2

140 1491/13 48 Male Cervical Lymph NodeLymphoma 2 3 3 3 3 2 0.94 2 3 3 3 3 3 1 2 3 3 2 3 3 0.88 2

141 1465/12 58 Male Parotid Salivary GlandWarthin's Tumor 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 3 2 2 3 2 0.76 2

142 2447/12 36 Male Sub-mandibular Salivary GlandChronic Sialadenitis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 2 0.65 2

143 2317/12 48 Male Parotid Salivary GlandSialadenosis 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 3 0.82 2

144 102/13 34 Female Parotid Salivary GlandPleomorphic Adenoma 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 3 3 2 3 3 0.88 2

145 146/13 65 Female Parotid Salivary GlandCarcinoma Of Parotid 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 2 0.76 2

146 1135/13 34 Female Parotid Salivary GlandMucoepidermoid Carcinoma 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 3 2 0.76 2

147 1854/13 57 Male Parotid Salivary GlandBenign Cystic Lesion-Parotid 1 2 3 3 3 2 0.82 1 2 3 3 3 2 0.82 1 2 3 2 3 3 0.82 1

148 1885/13 75 Male Parotid Salivary GlandCarcinoma Ex Pleomorphic Adenoma 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 3 2 3 3 0.82 1

149 1901/13 53 Male Parotid Salivary GlandAcute Inflammatory Process-Parotid 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 2 3 3 3 3 0.94 2

150 1963/13 47 Female Sub-mandibular Salivary GlandRetention Cyst-Submandibular Salivary Gland2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.82 2

MODIFIED ULTRAFAST PAPANICOLAOU STAIN RAPID ECONOMIC ACETIC ACID PAPANICOLAOU STAIN

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2 2 2 2 3 0.76 1 3 3 3 3 3 0.94

2 2 2 2 3 0.65 1 2 3 3 3 2 0.82

2 2 2 2 3 0.65 1 2 2 3 3 2 0.76

3 2 2 2 2 0.76 2 3 3 3 3 3 1

3 3 3 3 3 1 2 3 3 3 2 3 0.94

2 2 2 2 2 0.7 2 3 3 3 3 3 1

2 3 2 2 2 0.76 1 2 3 2 2 3 0.76

3 2 2 3 2 0.82 1 3 3 3 3 3 0.94

2 2 2 2 2 0.7 1 2 2 2 2 2 0.65

3 3 2 3 2 0.88 2 3 3 3 3 3 1

3 3 2 3 2 0.88 2 3 3 3 3 3 1

3 2 2 3 3 0.82 2 3 3 3 3 3 1

3 3 2 2 3 0.88 2 3 2 2 2 2 0.76

2 2 2 2 2 0.65 1 3 3 3 3 3 0.94

2 2 2 2 2 0.7 1 3 2 3 3 3 0.88

2 2 2 2 3 0.7 1 2 2 2 2 3 0.7

2 1 2 2 3 0.65 1 2 2 2 2 2 0.65

2 3 3 3 3 0.94 1 2 2 2 2 3 0.7

2 2 2 2 3 0.7 1 3 3 3 3 3 0.94

2 2 2 2 1 0.58 1 3 3 3 3 3 0.94

2 2 2 2 2 0.7 1 2 3 3 3 3 0.88

2 2 2 2 2 0.7 1 2 3 3 3 3 0.88

2 2 2 2 3 0.7 1 2 2 2 2 2 0.65

2 2 2 2 3 0.7 1 3 3 3 3 3 0.94

2 3 2 2 3 0.76 1 2 3 3 3 2 0.82

2 2 2 2 2 0.7 1 2 3 3 3 3 0.88

2 2 2 2 2 0.7 2 3 3 3 3 3 1

2 2 2 2 3 0.65 1 2 3 2 2 3 0.76

2 2 2 2 3 0.7 2 3 3 3 3 3 1

2 3 2 2 2 0.7 1 2 2 2 2 3 0.7

2 3 3 3 2 0.88 1 2 2 2 2 3 0.7

2 2 2 2 2 0.65 1 2 2 3 3 3 0.82

2 2 2 2 3 0.7 2 3 3 3 3 3 1

2 2 2 2 3 0.7 1 3 3 3 3 2 0.88

2 3 3 3 3 0.94 2 2 2 2 2 2 0.7

2 3 3 2 3 0.88 1 2 2 3 2 2 0.7

3 3 3 3 3 1 1 2 3 3 3 2 0.82

3 3 3 3 3 1 1 3 3 3 3 2 0.88

2 2 2 2 3 0.76 1 3 3 3 3 2 0.88

3 3 3 3 3 1 1 2 2 2 2 2 0.64

2 3 3 3 3 0.94 1 3 3 3 3 2 0.88

3 3 3 2 2 0.82 2 2 3 3 2 2 0.82

3 3 3 3 3 1 2 3 3 3 3 2 0.94

3 2 3 3 3 0.88 1 3 3 3 3 2 0.88

3 3 3 3 3 1 2 3 2 2 2 2 0.76

2 3 3 3 3 0.82 1 3 3 3 3 2 0.88

3 3 3 3 3 1 1 3 3 3 3 2 0.88

2 2 2 2 2 0.7 1 2 3 3 2 2 0.76

3 2 2 2 3 0.82 1 3 3 3 2 2 0.82

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

3 3 2 3 3 0.94 2 3 3 3 3 2 0.88

3 3 3 3 3 1 1 3 2 3 2 2 0.76

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

2 2 2 2 3 0.7 1 2 3 3 3 3 0.88

3 3 2 3 3 0.82 1 2 3 3 2 3 0.82

2 3 2 3 3 0.76 1 3 3 3 3 2 0.88

3 3 3 3 3 0.94 2 3 3 3 3 3 1

3 3 3 3 3 0.94 1 3 3 2 3 3 0.88

2 2 2 2 3 0.65 1 3 3 3 3 2 0.88

3 2 2 2 2 0.7 1 3 3 3 3 2 0.88

3 3 3 3 2 1 2 3 3 3 3 3 1

1 2 2 2 1 0.58 2 3 3 3 3 3 1

3 2 2 2 2 0.82 1 3 3 3 3 2 0.88

2 2 2 2 3 0.76 1 3 3 3 3 2 0.88

2 3 2 2 3 0.76 2 3 3 3 3 3 1

2 2 2 2 3 0.7 1 3 3 3 3 3 0.94

2 2 2 2 3 0.7 1 3 3 3 3 3 0.94

2 2 2 2 2 0.7 2 3 3 3 3 3 1

3 3 3 3 3 1 2 3 3 3 3 3 1

2 3 2 3 2 0.82 2 3 3 3 3 3 1

3 3 2 3 2 0.88 2 3 3 3 3 3 1

3 3 2 3 3 0.82 2 3 3 3 3 2 0.94

3 3 3 3 3 1 1 3 3 3 3 2 0.88

2 2 2 2 2 0.7 1 3 3 3 3 2 0.88

2 2 3 2 2 0.7 2 2 3 3 2 3 0.88

2 2 2 2 3 0.7 2 3 3 3 2 3 0.94

2 2 2 2 2 0.7 1 3 3 3 3 3 0.94

2 2 2 2 2 0.7 2 2 3 3 3 3 0.94

2 2 2 2 3 0.7 2 2 3 2 2 3 0.82

2 2 2 2 3 0.7 2 3 3 3 3 3 1

2 3 3 3 2 0.88 2 3 2 2 2 3 0.82

2 2 2 2 3 0.7 1 3 3 2 3 3 0.88

3 2 2 2 2 0.76 2 3 2 2 2 3 0.82

2 2 2 3 3 0.76 2 2 2 2 2 3 0.76

2 3 3 3 3 0.94 1 3 3 3 3 3 0.94

2 2 2 2 3 0.65 2 2 3 3 3 3 0.94

2 2 2 2 2 0.7 1 2 3 3 3 3 0.88

2 2 2 2 2 0.65 2 3 3 3 3 3 1

2 2 2 2 3 0.7 2 2 3 3 3 3 0.94

2 3 2 2 2 0.76 2 3 3 3 3 3 1

2 2 2 2 2 0.65 2 3 3 3 3 3 1

3 3 3 3 3 1 2 2 3 3 3 3 0.94

3 2 3 2 2 0.82 1 3 3 3 3 2 0.88

2 3 2 2 3 0.76 1 3 3 3 3 2 0.88

2 2 2 2 3 0.7 1 3 3 3 3 2 0.88

3 3 3 3 3 1 1 3 3 3 3 2 0.88

3 3 3 3 3 1 1 3 3 3 3 2 0.88

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

2 3 2 3 2 0.82 1 3 3 3 3 2 0.88

2 2 2 2 3 0.76 1 3 3 3 2 2 0.82

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

2 3 2 2 2 0.7 2 3 3 3 3 3 1

3 3 3 3 3 1 1 2 3 3 3 2 0.82

2 3 2 3 3 0.88 2 3 3 3 3 2 0.94

2 3 2 3 2 0.82 1 2 2 2 2 3 0.7

3 3 3 3 1 0.82 1 3 3 3 3 3 0.94

2 2 2 2 3 0.7 2 3 3 3 3 3 1

2 3 2 3 2 0.76 1 2 3 3 2 2 0.76

2 3 2 3 3 0.76 1 2 2 3 2 3 0.82

2 2 2 2 3 0.65 1 2 2 3 2 3 0.76

2 3 2 3 3 0.82 1 2 3 3 3 2 0.82

2 3 3 3 3 0.88 2 3 3 3 3 3 1

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

3 3 3 3 3 1 1 3 3 3 3 2 0.88

2 3 2 3 3 0.82 1 3 3 3 3 2 0.88

1 2 2 1 2 0.58 2 2 2 2 2 3 0.76

2 3 3 2 3 0.82 2 3 3 3 3 3 1

2 2 2 2 2 0.7 1 2 3 3 3 3 0.88

1 2 2 2 3 0.65 2 3 3 3 3 3 1

1 2 2 2 2 0.65 2 3 3 3 3 3 1

2 2 2 2 3 0.7 2 3 3 3 3 3 1

2 2 2 2 3 0.65 2 3 3 3 3 3 1

2 2 2 2 3 0.7 1 3 3 3 3 3 0.94

3 3 3 3 2 0.94 1 3 3 3 3 3 0.94

2 3 2 2 2 0.88 1 3 3 3 3 3 0.94

3 3 3 3 3 1 2 3 3 3 3 1 0.88

3 3 3 3 3 1 1 2 3 3 3 2 0.82

3 3 3 3 3 1 1 3 3 3 3 2 0.88

2 3 2 2 3 0.76 1 2 3 3 3 2 0.82

2 2 2 2 3 0.76 1 2 3 3 3 2 0.82

2 3 2 3 3 0.88 1 2 3 3 3 2 0.82

3 3 3 3 2 0.94 1 2 2 3 2 2 0.7

3 3 3 3 3 1 1 2 2 2 2 2 0.64

2 3 3 3 2 0.88 1 3 2 3 2 2 0.76

3 3 2 3 3 0.88 1 3 3 3 3 2 0.88

3 3 2 3 3 0.94 1 3 3 3 3 2 0.88

3 3 3 3 3 0.94 2 3 3 3 3 3 1

3 3 2 3 2 0.88 2 3 3 3 3 3 1

3 3 2 3 2 0.88 1 3 3 3 3 2 0.88

3 2 2 3 2 0.82 2 3 3 3 3 3 1

2 2 2 2 2 0.7 2 3 3 3 3 3 1

2 2 3 2 2 0.76 1 2 2 3 3 2 0.76

3 3 2 3 3 0.94 1 3 3 3 3 3 0.94

3 3 2 2 3 0.82 1 2 2 2 2 2 0.65

2 2 2 2 2 0.76 1 2 3 3 2 2 0.76

2 2 2 2 3 0.7 1 2 2 2 3 2 0.7

2 3 2 3 3 0.82 1 2 3 3 3 2 0.82

3 3 2 3 3 0.94 2 3 3 3 3 2 0.94

3 3 2 3 3 0.94 2 3 3 3 3 2 0.94

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