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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
24 SQUAMOUS CELL CARCINOMA SECONDARIES
IN LYMPH NODE, MGG STAIN 67
25 SQUAMOUS CELL CARCINOMA SECONDARIES
IN LYMPH NODE, REAP STAIN 68
26 SQUAMOUS CELL CARCINOMA SECONDARIES
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
H & E, PAP, MGG, MUFP and REAP 54
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
H & E, PAP, MGG, MUFP and REAP 54
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
9. 95% Ethanol 6 slow dips
10. 100% Ethanol 6 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
1% acetic acid 10 dips
OG-6 10 dips
1% acetic acid 10 dips
EA-50 10 dips
1% acetic acid 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%)
Nuclear characteristics
• 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%)
Air drying artifacts
• >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
• Poorly preserved 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
• 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%)
Nuclear characteristics
• Smudgy Chromatin 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
• 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%)
Air drying artifacts
• >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
• Poorly preserved 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
• Moderately
preserved 0(0%) 0(0%) 2(20%) 5(50%) 3(30%)
• Well preserved 10(100%) 10(100%) 8(80%) 5(50%) 7(70%)
Nuclear characteristics
• Smudgy Chromatin 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
• 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%)
Air drying artifacts
• >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
For H&E Stain, Quality index score difference is statistically significant (p value
Mean Quality index score for H&E stain is maximum for breast followed by thyroid,
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
For H&E Stain, Quality index score difference is statistically significant (p value
Mean Quality index score for H&E stain is maximum for breast followed by thyroid,
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
P=0.397 by Fisher Exact test
associated with P value of 0.397.
Mean Quality index score for Pap stain is maximum for breast and thyroid followed
node and salivary gland.
GRAPH 4. PAP stain in different organs
Lymph Node
Breast Salivary Gland
0.81-1.0
<0.80
PAP Score
45
Total
2(1.3%)
148(98.7%)
150(100%)
0.95±0.05
Mean Quality index score for Pap stain is maximum for breast and thyroid followed
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
P=0.830 by Fisher Exact test
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
P=0.830 by Fisher Exact test
Mean Quality index score for MGG stain is maximum for breast,thyroid and lymph
node followed by salivary gland.
GRAPH 5. MGG stain in different organs
Lymph Node
Breast Salivary Gland
0.81-1.0
<0.80
MGG Score
46
Total
68(45.3%)
82(54.7%)
150(100%)
0.80±0.09
Mean Quality index score for MGG stain is maximum for breast,thyroid and lymph
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
P=0.577 by Fisher Exact test
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
Mean Quality index score for MUFP stain is maximum for lymph node followed by
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 Gill’s hematoxylin in MUFP and
GRAPH 6. MUFP stain in different organs:
Lymph Node
Breast Salivary Gland
0.81-1.0
<0.80
MUFP Score
47
Total
75(50%)
75(50%)
150(100%)
0.82±0.12
Mean Quality index score for MUFP stain is maximum for lymph node followed by
Nuclear stain used in MUFP is Gill’s hematoxylin. For 10 cases of FNAC smears,
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
P=0.007 by Fisher Exact test
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
P=0.007 by Fisher Exact test
For REAP stain, score difference is statistically significant and more associated with
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
Lymph Node
Breast Salivary Gland
0.81-1.0
<0.80
REAP Score
48
Total
29(19.3%)
121(80.7%)
150(100%)
0.88±0.09
associated with
Mean Quality index score for REAP stain is maximum for breast followed by lymph
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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%)
Value P value Difference is
250 <0.001 Significant
50
of Overall staining in H & E, PAP, MGG, MUFP and REAP
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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%)
Value P value Difference is
127 <0.001 Significant
51
Correlation of Cell Morphology in H & E, PAP, MGG, MUFP and REAP
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, MUFP and REAP
Based on total number of units , 600 samples are considered for statistical significance
Correlation of nuclear characteristics in H & E, PAP, MGG,
and REAP
PAP MGG MUFP REAP
crisp
Moderately Crisp
Smudgy
Nuclear characteristics
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%)
Value P value Difference is
251 <0.001 Significant
52
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
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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
Based on total number of units , 600 samples are considered for significance
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%)
Value P value Difference is
1.47 0.690 Not Significant
54
Correlation of air drying artifacts in H & E, PAP, MGG, MUFP and REAP
Based on total number of units , 600 samples are considered for significance
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
FIGURE 12. CARCINOMA OF BREAST, Pap stain, 100x and 400x
61
FIBROADENOMA OF BREAST, MUFP stain, 100X
FIGURE 12. CARCINOMA OF BREAST, Pap stain, 100x and 400x
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
Nuclear characteristics
Cytoplasmic details
Air drying artifacts
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
SE
RIA
L N
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LOG
Y N
UM
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AG
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GA
N
IMP
RE
SSIO
N
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CK
GR
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OV
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ALL
ST
AIN
ING
CE
LL M
OR
PH
OLO
GY
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AR
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TE
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TO
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IC D
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G A
RT
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IND
EX
BA
CK
GR
OU
ND
OV
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ST
AIN
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LL M
OR
PH
OLO
GY
NU
CLE
AR
CH
AR
AC
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RIS
TIC
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PLA
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IC D
ET
AIL
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G A
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IFA
CT
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TY
IN
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X
BA
CK
GR
OU
ND
OV
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ALL
ST
AIN
ING
CE
LL M
OR
PH
OLO
GY
NU
CLE
AR
CH
AR
AC
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RIS
TIC
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PLA
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IC D
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AIL
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CK
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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
2 1369/12 24 Female Thyroid Nodular Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 2 2 2 2 0.65 1
3 1372/12 76 Female Thyroid Nodular Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 1 2 2 2 1 0.52 1
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
8 2193/12 31 Female Thyroid Nodular Goiter 1 3 3 2 3 2 0.82 1 3 3 3 3 3 0.94 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
11 2264/12 50 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 3 3 2 3 2 0.82 2
12 2272/12 30 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 3 3 0.76 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
16 2481/12 36 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 3 3 3 3 3 0.88 2
17 2491/12 30 Female Thyroid Nodular Goiter 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
18 2510/12 35 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 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
21 2513/12 22 Female Thyroid Nodular Goiter 2 3 2 2 2 3 0.82 2 3 3 3 3 3 1 1 3 3 3 3 2 0.88 2
22 2517/12 30 Female Thyroid Lymphocytic Thyroiditis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 2 2 0.7 2
23 2529/12 25 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2
24 2592/12 50 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 3 3 3 3 0.94 2
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
26 2610/12 37 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 2 2 0.76 2
27 2614/12 40 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 2 0.65 2
28 2650/12 53 Female 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
29 2655/12 8 Female Thyroid Colloid Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 3 3 3 0.88 2
30 2684/12 29 Female Thyroid Nodular Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 2 2 2 3 0.76 1
31 2685/12 53 Female Thyroid Colloid Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 2 0.82 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
33 2705/12 54 Female Thyroid Nodular Goiter 1 3 3 3 3 3 0.94 2 3 3 3 3 3 1 1 2 2 3 3 3 0.76 2
34 59/13 33 Female Thyroid Nodular Goiter 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
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
37 181/13 60 Female Thyroid Colloid Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 3 2 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
39 257/13 35 Female Thyroid Nodular Goiter 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
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
41 277/13 52 Female Thyroid Nodular 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 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
43 345/13 34 Female Thyroid Colloid Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 2 3 3 3 3 0.88 2
44 434/13 50 Female Thyroid Nodular Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 2 3 3 3 0.82 2
45 453/13 32 Female Thyroid Colloid Goiter 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2
46 470/13 45 Female Thyroid Lymphocytic Thyroiditis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 3 2 3 0.88 1
47 1036/13 38 Female Thyroid Hashimoto's Thyroiditis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.76 2
48 1051/13 33 Female Thyroid Hashimoto's Thyroiditis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 2 3 3 0.82 2
49 1083/13 32 Female Thyroid Hashimoto's Thyroiditis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 2 2 3 3 0.76 2
50 1085/13 24 Female Thyroid Hashimoto's Thyroiditis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 3 2 3 3 0.82 2
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
52 1110/13 28 Female Thyroid Nodular Goiter 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 3 2 3 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
54 2709/12 55 Female Thyroid Lymphocytic Thyroiditis 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2
55 1316/13 45 Female Thyroid Colloid Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 2 0.88 1 2 3 2 2 3 0.7 1
56 1317/13 36 Female Thyroid Colloid Goiter 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
57 1364/13 35 Female Thyroid Colloid Goiter 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 2 2 3 0.76 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
59 1419/13 38 Female Thyroid Colloid Goiter 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 2 3 2 2 3 0.7 1
60 1427/13 45 Female Thyroid Lymphocytic Thyroiditis 1 3 3 3 3 3 0.94 1 3 3 3 3 3 0.94 1 3 2 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
66 2134/12 42 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
69 2190/12 46 Female Breast Fibroadenoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 3 3 0.82 2
70 2242/12 22 Female Breast Fibroadenoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 3 2 3 3 0.88 2
71 2243/12 47 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 2 2 3 2 0.82 1
72 2271/12 45 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 3 3 2 3 2 0.88 2
73 2316/12 50 Female Breast Carcinoma Of Breast 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 3 2 2 0.82 2
74 2326/12 50 Female Breast Carcinoma Of Breast 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 2 2 2 3 0.7 2
75 2341/12 55 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 2 2 3 0.65 1
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
79 2390/12 40 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
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
82 2486/12 50 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 2 3 3 2 0.76 1
83 2487/12 40 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 3 3 3 2 0.88 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
87 2554/12 44 Female Breast Fibrocystic Disease 2 3 3 3 3 3 1 2 3 3 3 3 3 1 2 2 2 2 2 2 0.7 2
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
90 2659/12 50 Female Breast Fibroadenoma 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 3 2 2 0.76 2
91 2704/12 32 Female Breast Carcinoma Of Breast 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 3 3 3 3 2 0.88 1
92 2746/12 40 Female Breast Acute Suppurative Process 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 3 2 0.76 2
93 85/13 32 Female Breast Fibroadenoma 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 2 2 3 0.82 2
94 192/13 44 Female Breast Fibroadenoma 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 2 2 2 3 0.7 2
95 193/13 66 Female Breast Carcinoma Of Breast 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 3 3 3 3 0.88 1
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
98 471/13 21 Female Breast Fibroadenoma 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 2 3 2 3 0.76 2
99 1055/13 40 Female Breast Fibroadenoma 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
100 1077/13 49 Female Breast Carcinoma Of Breast 1 3 3 3 3 2 0.88 2 3 3 3 3 2 0.94 1 2 3 2 3 3 0.82 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
102 1264/13 65 Female Breast Carcinoma Of Breast 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 3 3 3 2 3 0.88 2
103 1321/13 34 Female Breast Acute Suppurative Process 2 3 3 3 3 3 1 2 3 3 3 3 3 1 1 2 3 2 2 3 0.7 1
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
114 2275/12 40 Female Cervical Lymph NodeTuberculous Lymphadenitis 1 3 3 3 3 2 0.88 1 3 3 3 3 2 0.88 1 2 3 3 2 3 0.82 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
116 2314/12 40 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 3 3 3 2 3 0.82 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
119 2373/12 35 Male Cervical Lymph NodeTuberculous Lymphadenitis 1 2 2 2 2 3 0.7 2 2 3 3 3 3 0.94 1 2 2 2 2 2 0.65 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
128 118/13 35 Male Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 2 2 3 2 3 3 0.82 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
131 202/13 25 Female Cervical Lymph NodeTuberculous Lymphadenitis 2 3 3 3 3 2 0.94 2 3 3 3 3 2 0.94 1 2 2 2 2 3 0.7 2
132 239/13 24 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 3 3 0.94 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
OV
ER
ALL
ST
AIN
ING
CE
LL M
OR
PH
OLO
GY
NU
CLE
AR
CH
AR
AC
TE
RIS
TIC
S
CY
TO
PLA
SM
IC F
EA
TU
RE
S
AIR
DR
YIN
G A
RT
IFA
CT
S
QU
ALI
TY
IN
DE
X
BA
CK
GR
OU
ND
OV
ER
ALL
ST
AIN
ING
CE
LL M
OR
PH
OLO
GY
NU
CLE
AR
CH
AR
AC
TE
RIS
TIC
S
CY
TO
PLA
SM
IC F
EA
TU
RE
S
AIR
DR
YIN
G A
RT
IFA
CT
S
QU
ALI
TY
IN
DE
X
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