COMPARATIVE EVALUATION OF CELL BLOCK AND SMEAR

SAMPLING TECHNIQUE IN FINE NEEDLE ASPIRATION CYTOLOGY

A SEMINAR PRESENTED BY

OWUDA BENEDICT OFUJE

BMS 1501946

PRESENTED TO THE DEPARTMENT OF MEDICAL LABORATORY

SCIENCE, FACULTY OF BASIC MEDICAL SCIENCES, IN PARTIAL
FULFILLMENT OF THE REQUIREMENT FOR AWARD OF BACHELOR
OF MEDICAL LABORATORY SCIENCES (BMLS) OF UNIVERSITY OF
BENIN-CITY, EDO STATE, NIGERIA.

SUPERVISOR:

DR. EFOSA BOLAJI ODIGIE

JUNE, 2021.
 CERTIFICATION
This is to certify that this work was carried out by OWUDA BENEDICT OFUJE
with matriculation number BMS1501946 under the supervision of DR EFOSA
BOLAJI ODIGIE and was submitted to the Department of Medical Laboratory
Science, Faculty of Basic Medical Science, University of Benin, in partial fulfillment
of the requirement for the Award of Bachelor of Basic Medical Sciences (BMLS) of
University of Benin-City, Edo State, Nigeria.

_________________________ ____________________

DR. EFOSA BOLAJI ODIGIE DATE

(SUPERVISOR)

______________________ ___________________
PROF.(MRS) E.O. OSIME DATE

(HEAD OF DEPARTMENT)

_______________________ ___________________
EXTERNAL SUPERVISOR DATE

ii
 DEDICATION

This work is dedicated to God Almighty who has given me grace and shown me
mercy and favour in my academics, and to my parents who has backed me up in my
studies with Physical and Spiritual support.

iii
 AKNOWLEDGEMENTS

My earnest gratitude goes to God the creator of heaven and the earth who has kept me
alive till this very moment. I also wish to express my profound gratitude to my
supervisor, Dr. Efosa Bolaji Odigie for his tolerance and persistence in ensuring that
this work is completed on time.

My sincere appreciation goes to my parents, Mr. and Mrs. Job Owuda who has been
there for me financially and otherwise. I also want to express my sincere gratitude to
the person of Mr. Kelvin Odega whose immense contribution makes this work less
burdensome and the finally to all of my siblings who contribute one way or the other
in ensuring the success of this work. May God reward you in all your ambition and
grant you all your heart desires.

iv
 TABLE OF CONTENTS
CERTIFICATION........................................................................................................... ii

DEDICATION ...............................................................................................................iii

AKNOWLEDGEMENTS .............................................................................................. iv

LIST OF FIGURES......................................................................................................viii

LIST OF PLATES........................................................................................................viii

ABSTRACT ................................................................................................................... ix

CHAPTER ONE: INTRODUCTION ......................................................................... 1

1.1 BACKGROUND OF STUDY ............................................................................... 1

1.2 STATEMENT OF PROBLEMS ........................................................................... 2

1.4 SIGNIFICANCE OF STUDY ............................................................................... 4

1.5 AIM OF STUDY ................................................................................................... 4

1.6 SPECIFIC OBJECTIVES ...................................................................................... 4

1.7 RESEARCH QUESTIONS ................................................................................... 5

CHAPTER TWO: LITERATURE REVIEW ............................................................ 6

2.1 CELL BLOCK PREPARATION ....................................................................... 6

2.1.2 USEFULNESS OF CELL BLOCK PREPARATION .................................... 8

2.1.3 ADVANTAGES OF CELL BLOCK TECHNIQUE ......................................... 8

2.1.4 LIMITATIONS OF CELL BLOCK TECHNIQUE ......................................... 11

2.1.5 MODIFICATION OF CELL BLOCK TECHNIQUE ..................................... 11

2.2 CELL BLOCK AND SMEAR CYTOLOGY .................................................. 12

2.3 FINE NEEDLE ASPIRATION CYTOLOGY .................................................... 16

2.4.0 FINE NEEDLE ASPIRATION CYTOLOGY SAMPLES .......................... 17

v
 2.4.1 BREAST FNAC ........................................................................................... 17

2.4.2 LIVER FNAC ................................................................................................... 22

2.4.3 ORAL AND SALIVARY GLAND LESIONS FNAC ................................. 22

2.4.4 FNAC OF SOFT TISSUE LESIONS AND TUMORS ............................... 24

2.5 ROLE OF CELL BLOCK IN FNAC SAMPLE PREPARATION ................. 26

2.6.0 SMEAR SAMPLING TECHNIQUES IN FNAC......................................... 28

2.7 LIMITATIONS OF CONVENTIONAL FINE NEEDLE ASPIRATION

SMEAR ...................................................................................................................... 31

CHAPTER THREE: MATERIALS AND METHOD ............................................ 33

3.1 MATERIALS ....................................................................................................... 33

3.1.1 Histogel ............................................................................................................. 33

3.1.2 Liquid based cytology kit.................................................................................. 33

3.2 METHODOLOGY............................................................................................... 33

3.2.1 Sample Collection for Conventional Staining .................................................. 33

3.2.2 Sample Preparation for Liquid based Cytology Staining ................................. 34

3.2.3 Rapid Romanowsky Stain ................................................................................. 34

3.2.4 Hematoxylin and Eosin staining ....................................................................... 35

3.2.5 Papinicolaou Staining ....................................................................................... 36

3.2.6 Cell Block Processsing ..................................................................................... 37

3.2.7 Staining Procedure for Cell Block sections ...................................................... 38

3.3. ETHICAL APPROVAL ..................................................................................... 40

3.4 LOCATION OF STUDY ..................................................................................... 41

CHAPTER FOUR: RESULTS .................................................................................. 42

CHAPTER FIVE: DISCUSSION, SUMMARY AND CONCLUSION ................ 52

vi
 5.1 DISCUSSION ...................................................................................................... 52

5.2 SUMMARY ......................................................................................................... 55

5.3 CONCLUSION .................................................................................................... 56

5.4 RECOMMENDATION ....................................................................................... 56

REFERENCES .............................................................................................................. 57

vii
 LIST OF FIGURES
Figure i: Cell block Preparation method

Figure ii: Demonstrating Link between cytology and Histopathology with cell

block

Figure iii: Comparing Cytosmears and Cell block

Figure iv: Demonstrating Breast and Thyroid FNAC

Figure v: Thin Prep Smear

LIST OF PLATES
Plate 1: Ascitic fluid for cell block. Conventional smears, liquid based cytology

Plate 2: Ascitic fluid for cell block, conventional smears and liquid based cytology.

Plate 3: Pleural fluid for Cell block, Conventional smear and Liquid based Cytology

Plate 4: Breast Cyst for Cell block, conventional and Liquid based Cytology.

viii
 ABSTRACT
This study comparatively analyzed diagnostic effectiveness of stained sections of cell
block prepared from non-gynecological samples, which include mainly FNAC and
stained smears prepared simultaneously from same samples each; utilizing routine
smear sampling techniques, which include both liquid-based cytology and
conventional methods respectively. Among other objectives, this study analyzed
which of the various methods that can further improve a high standard cytological
screening, and provided solutions to the problem encountered when FNA does not
yield sufficient information for precise diagnosis and also pin point the risk of false
negative or intermediate diagnosis that may exist with respect to the various methods
by comparatively processing a number of samples and making examination with three
staining technique; the Rapid romanowsky stain, the Pap stain and H/E staining for
both smears of the Liquid base and conventional method and sections of the Cell block
technique prepared from the same samples. Among others, Results from Breast cyst
FNAC as a case study shows features with (LBC) such as; scanty clusters of
cytological normal polygonal to spindle shaped cells mixed with infiltrates of
neutrophils in a clear neat background which were not demonstrated with the Cell
block and Conventional method. While with Pleural Fluid Cell block, and LBC
reveals similar features some of which were not demonstrated in a conventional smear
but all suggestive of a benign smear. Therefore, there is profound improvement in
diagnostic cytology with the simultaneous utilization of this 3 technique with Cell
block giving a higher level of clarified diagnosis for suspicious lesions.

ix
 CHAPTER ONE
INTRODUCTION

1.1 BACKGROUND OF STUDY

Cell block technique is a method that allows cytological specimens ranging from

microscopic to grossly visible tissue fragments to be processed into paraffin blocks

through which the routine histopathology sectioning and staining can be carried out.

Cell block can be used in providing necessary support for molecular and

immunocytochemistry studies (Krogerus et al., 2018). On the other hand, Liquid-

based cytology is a common technique used in the cytology lab for basic sample

preparation. This technique was first tried on Pap smear after being introduced and

then finally gained approval from the Food and Drug Association (FDA) in 1996

(Koss et al., 2005). Liquid-based cytology is performed not only for gynecological

cytology but also for non-gynecological cytology such as the conventional fine needle

aspiration cytology (FNAC), guided Fine Needle aspiration cytology and some other

fluid cytology.

As at the time of writing this project, there has not been a recent standardized method

for creating cell block from a Fine Needle aspirate. The varying methods are still

presently in use by different Histopathology laboratories (Nigro et al., 2007). The

major contrast in the various method as concerned to the three standard stages in the

cell block preparation can be observed as in the liquid medium for clearing the

specimen material out of the needle and hub, in the preservative utilized for sample

1
fixation and also in the process utilized for the formation the clot for the Cell block in

the laboratory. Various special fluids employed for rising the FNA needle, include

saline, cell culture solutions such as RPMI or Hank balanced salt solution, and

fixatives such as Cytolyt or formalin. The common clotting agents are plasma and

thrombin, agar, and Histogel (HG). The collodion bag (ColB) technique is also another

choice of clotting agent (Krogerus et al., 2018).

Sequel to an undesired result or low quality output, Pap smear testing has been

considered to have some limitation. This condition arises not only as a result of the

reduced standard of sample preparation but also because artifacts, blood, inflammation,

bad cell fixation, and inhomogeneous distribution of cells acts on blotting out the true

smear appearance which may serve as a source of errors result interpretation. This is

the reason why Liquid-based cytology was brought out as the major second choice of

cytological analysis. In the liquid-based cytology technique, the cells are obtained

using the common sampling material and then it is rinsed into a bottle with

preservative fluid instead of smearing it directly on a slide (Arbyn et al., 2007). Since

just a part of a whole bulk sample is required for the test, the remaining material in the

vial can be utilized for other type of testing (Arbyn et al., 2004).

1.2 STATEMENT OF PROBLEMS

The major challenge with cell block when the Histogel method is utilized is that the

pellet formed with this method has a low level of gross macroscopic appearance in the

2
Paraffin block. Owing to this the correct level of sectioning during microtomy is

prevented (Ronald et al., 2016). The level of unreliability in the laboratory had been a

serious cause for concern for several years, remaining incredibly high despite repeated

efforts to review or redefined the reporting standard in the laboratory. The major

reasons why conventional smears are being reported as unsatisfactory in the laboratory

is because of the obscuration caused by scanty cellular material, purulent exudate,

cytolysis, or air-drying artifact (Reetika et al, 2020). However, with the advent of

modern improvement on laboratory techniques for cytological analysis, all these

problems are now rare or absent with LBC, the most frequent cause of unsatisfactory

smears now being insufficient cellular material for assessment (Reetika et al, 2020).

1.3 JUSTIFICATION OF STUDY

In Routine laboratory practice, the quest for an error free result on cytological

diagnosis is an indispensable factor for consideration. Research proves that there is

quite an arithmetic increase in the level of false negative and false positive diagnostic

result on cytology samples when the use of certain cytology based techniques are

employed. Patient also experience some discomfort and inconveniences with the use

of unreliable control method. For this reason, this study provides a demand for a more

reliable technique through which several diagnoses can be carried out from a single

sample of fine Needle aspirate and also give room to assess the level of errors in

3
cytological diagnosis that can occur through a comparative analysis of the

effectiveness of the Cell block technique and Smear sampling technique.

1.4 SIGNIFICANCE OF STUDY

The Significance of this Study is targeted at optimizing the standard of laboratory

techniques that enhance the analysis of Fine Needle Aspirate samples in this current

trend of Laboratory science where Precision and accuracy as well as a less invasive

diagnostic method is required for a good Cytological analysis.

1.5 AIM OF STUDY

The aim of this study is to comparatively analyse the effectiveness of the Cell Block

and the Smear sampling technique in Fine Needle Aspiration Cytology.

1.6 SPECIFIC OBJECTIVES

The Specific objective were to:
1. Compare specimen adequacy and diagnostic agreement between Cell block and
Smear sampling technique in Fine Needle Aspiration Cytology.
2. Evaluate whether Cell block technique can improve high‐standard Fine Needle
Aspiration Cytology screening further.
3. Determine the technique that gives better quality of cell microscopic appearance.
4. Assess the technique that is of tremendous advantage in Preserving Fine Needle
aspirate for Future Use.

4
1.7 RESEARCH QUESTIONS
Is there more yield of cells in the Smear sampling technique than the Cell block

technique?

What method can be more accurate?

Which method gives a high chance of cell storage?

Which method provide gives a better quality of cell microscopic appearance?

Which method functions at low cost?

5
 CHAPTER TWO
2.0 LITERATURE REVIEW

2.1 CELL BLOCK PREPARATION

The production of a good cell block of sufficient quality plays a very great role of

advantage in the diagnostic usefulness of cytology samples. FNAC Samples and some

concentrated fluid samples are commonly processed into Cell block. The Preservation

of residual samples of residual samples with the sole aim of producing cell block is a

common regular practice especially when the cytopathologist envisage the need of

some special cytological studies such as molecular and immuno-cytochemical and

Histochemical studies (Kristin et al., 2017).

6
Figure i: Cell block Preparation Method (Anam, 2017).

7
2.1.2 USEFULNESS OF CELL BLOCK PREPARATION
The usefulness of cell block preparation in cytopathological diagnosis conclusively

have a high level of significance, because it gives room for several special

investigations and in addition a more improved cytological diagnosis (Shehnaz et al.,

2012).

Cell-block preparations obtained from cell sediment can serve as a useful aid to the

routine cytological methods used for pleural and peritoneal fluids (Geethu et al., 2015).

Cell blocks prepared from residual tissue fluids and fine-needle aspirations can be

useful adjuncts to smears for establishing a more definitive cyto-pathologic diagnosis.

They can be particularly useful for categorization of tumors that otherwise may not be

possible from smears themselves. It also plays an important role when there is a need

for special stains or immunohistochemistry. There are many studies done to compare

the usefulness of cell blocks with that of smears in fine needle aspiration materials, but

only a few in the case of serous fluids. In this context the present study has been

undertaken to assess the utility of the cell block preparation method in increasing the

sensitivity of cyto-diagnosis of serous fluids (Geethu et al., 2015).

2.1.3 ADVANTAGES OF CELL BLOCK TECHNIQUE

This technique uses the retrieval of small fragments of tissue from the Sample aspirate

for processing into paraffin blocks.

8
The cell block technique employs the retrieval of small tissue fragments from a FNA

specimen which are processed to form a paraffin block. It is established method

known for its efficient diagnostic accuracy and cellular yield. The availability of

sufficient tissue sections gives room for multiple immunostains and other studies to be

performed in a similar pattern to paraffin sections produced in histopathology

(Shehnaz et al., 2012).

9
Figure ii. Demonstrating Link between cytology and Histopathology with cell
block (Anam, 2017).

10
2.1.4 LIMITATIONS OF CELL BLOCK TECHNIQUE
A delay in immersing the cell block specimen into fixative immediately after

collection and variation in FNA technique may result in the deterioration of cells in

the cell block specimen.

This variation in technique determines how well an adequate cell block samples is

collected. This is also dependent on the experience and skills of the physician

collecting the Fine needle aspirates and the high cellularity of the aspirates (Shehnaz et

al., 2012).

2.1.5 MODIFICATION OF CELL BLOCK TECHNIQUE

Direct FNA smears and cell blocks are simultaneously required for the diagnostic

work-up of patients because they complement each other. The former to assess

morphology, and the latter for optimal immune cytochemistry results. With the

consideration of limitation of resources, the cost implications of performing both

conventional and blocked smears on all FNA material gives allowance for a

subsequent evaluation.

However Fine needle aspiration techniques can be modified or improved on by

examining the use of 10% neutral buffered formalin (NBF) as the fixative of choice in

preparing cell block samples for immunohistochemistry (IHC), and reducing the time

lapse between sample collection and fixation and standardization of FNA technique

among personnel (Shehnaz et al., 2012).

11
2.2 CELL BLOCK AND SMEAR CYTOLOGY
Cytological analysis serves as a unique way of examining the body cavity fluids for

the presence or absence of Malignancy. Several researches shows that the diagnosis or

detection of malignancy is based on Fluid examination. The result of this diagnosis is

hinged on the nature of the disease and the degree of the malignancy. Cytological

analysis serves as a unique way of examining the body cavity fluid for the presence or

absence of malignancy. Several research shows that the diagnosis or detection of

malignancy is mainly based on fluid examination. The result of this diagnosis is

hinged on the nature of the disease and the degree of malignancy. Cytological

evaluation of fluid samples of patient can be done in different ways in the laboratories.

Majority of the laboratories employ the method of routine cytological smears for this

purpose. A research work conducted in 1996 shows a percentage correlation in the

parameters of efficiency, specificity and sensitivity in 4297fluid samples to be 98.7%

95.7%, 44.5% (Oyafuso et al., 1996). Another result not too different from this was

also shown to prove this from a research conducted in 1999 (Motherby et al., 1999).

These research results actually imply that the accuracy of diagnosis in effusion

cytology through the use of routine smears is unsatisfactory and should be improved.

This is why employing various adjuvant technique should be recommended.

The use of cell-block as a technique has not only been on trend for years but has also

received an approved level of acceptance. A result from a research study on effusions

that was carried out in 2005 shows that there are about 120 cases of different reactive

12
effusion which sum up to 63.15% and 70 cases of malignant effusion which sum up to

(36.85%) out of 190 cases studied (Meenu et al., 2005). It was also discovered from

the study out of the 120 cases of reactive effusion, the Proportion of Pericardial,

Pleural and peritoneal effusion were 6.7 %, 48.3 % and 45% respectively. It was also

shown that 18.33% of cases was found to arise as a result of tuberculosis. From the

research, the level of reactive and malignant effusions was closely equal. Tuberculosis

remains the most common cause of reactive effusion. This perhaps is owing to the

high prevalence of tuberculosis and also based on the fact that majority of the pleural

fluid samples are sent from Regional Institute of Chest Diseases where a large number

of TB patients are admitted.

Adenocarcinoma is known to be the most common cyto-pathologic diagnosis rendered

in malignant pleural effusions from research. 50% of the diagnostic result was shown

to be composed of Primary adenocarcinoma of the lungs.

Non-small cell carcinoma was the interpretation of the diagnostic result sequel to the

fact that the differentiation was not clear in 3 Occasions. In some other cases, though

the Primary site could not be identified yet adenocarcinoma was confirmed.

Lymphomatous involvement was seen in 5 cases. Primary site of tumor was identified

as breast in 7 cases. An exceptional cause of malignant effusion was also seen to arise

from both the small cell carcinoma and the squamous cell carcinoma because it was

discovered and identified on 3 occasions.

13
The carcinoma of the ovary as well as Adenocarcinoma of gastrointestinal tract is

attributed as the major cause of malignant ascites.

From a comparative study between Cell block and routine cytology smears on reactive

effusions, results show that from 75 samples of malignant

effusions, 21(28%) samples were accounted to be positive for malignant cells by

routine cytological smears. While with the cell-block technique diagnosis of 47 cases

(63%) of malignant cells was given (Thaper et al., 2005). The specificity of smears

and cell blocks in the detection of malignancy is considerably high. From studies it has

be proven that the sensitivity of smears and cell-blocks shows some percentage level

of correlation but has a reduced percentage level when it has to do with the detection

of malignancy using smears (Nithyananda et al., 2000). This therefore prove that

employing cell-block technique has a high level of efficiency in diagnosis.

14
Figure iii. Comparing Cytosmears and Cell block (Anam, 2017).

15
2.3 FINE NEEDLE ASPIRATION CYTOLOGY
Fine needle aspiration cytology (FNAC) is a procedure to obtain cells and tissue

fragments through a needle introduced into abnormal tissue or mass and its cytological

study (Shipra et al., 2018). It is a technique through which samples collected from a

lesion with the aid of a thin bore needle and are processed for cytopathological

diagnosis. It is applied as a means for clinical detection of liver disease, subcutaneous

soft tissue mass, salivary gland diseases, oral diseases, breast lumps, and thyroid

nodules.

The history of Fine Needle aspiration cytology is dated back to be introduced in the

1930s by Martin, Ellis and Stewart (Shipra et al., 2018). It was known as an

acceptable cytology technique in the late 1950s (Babu, 2013).

FNAC samples is mainly aspirated from common palpable body mass lesions such as

the, pelvic organs, bone, breast, testicles, Palpable abdominal lesions, prostate & joint

spaces, lungs, retro peritoneum etc (Babu, 2013).

Fine needle aspiration incorporates four sequential methods which include Palpation,

aspiration, smear preparation, Microscopy. Aspiration of samples using these methods

is based on the principle of negative pressure present on the syringe holding the tissue

against the sharp cutting edge of the needle. Fine needle aspiration cytology is not

required for cases of Pancreatitis, emphysema, or bleeding. Sample aspiration requires

clinical skills which varies from familiarity with the general anatomy (Babu, 2013).

16
For aspiration of specimens, the FNAC basically requires a sterile syringe and needle,

the length of the needle depending on the location of the organ to be sampled. The

needle thickness is usually in the range of 0.5 -0.9mm. A special handle can be

attached to the syringe to allow single hand grip, freeing the other hand for palpation

and fixation of the mass if it is mobile (Ochie et al., 2007).

2.4.0 FINE NEEDLE ASPIRATION CYTOLOGY SAMPLES

2.4.1 BREAST FNAC

Fine-needle aspiration cytology (FNAC) is an established, well defined, cost-effective

and accurate, method for diagnosing lesions in different organs, including the breast.

The method has a low level of invasiveness and side effects (Aasmund et al., 2011).

The technique for the preoperative detection of the carcinoma of the breast is highly

based on the use of fine-needle aspiration cytology (FNAC).

Breast cancer is the highly trending form of cancer in Indian women, having taken

predominance over cervical cancer (Gupta, 2016).

Fine-needle aspiration cytology (FNAC) is widely accepted as a rapid, reliable,

secured diagnostic technique for distinguishing non-neoplastic from neoplastic breast

lesions.

The cases of correct diagnosis of breast cancer is in 99% based on the combination of

clinical examination, mammography, and simple, non-invasive, cost-effective

outpatient department methods using fine-needle aspiration cytology (FNAC). FNAC

17
techniques plays a great role of usefulness for tumors which are easily accessible for

palpation (Nasar et al., 2011).

Globally, among female cancers, breast cancer has become a more prevailing cancer

accounting for about a quarter of all cancers having approximated recent cancer cases

of 150,000 diagnosed in 2016. Women from less developed regions have a quite large

number of cases compared to other women from the more developed regions (Malvia

et al., 2017).

Report on FNAC is of great value because it provides the required information for the

management of patients, in order to proceed with more invasive diagnostic methods or

surgical treatment, and to decide what form of operation to perform. FNAC play a

major role in the preoperative phase, both for palpable and non-palpable lesions, using

ultrasound or stereotactic guidance (Zagorianakou et al., 2005).

Patients are at a disadvantage as a result of economical restrictions, low budget for

health care and screening programmes, and mostly due to the high cost of

sophisticated diagnostic methods in developing countries. This is why FNAC should

be employed as a routine diagnostic method because of its low cost compared with the

others and this policy makes health care to women with breast cancer highly

accessible and procurable (Zagorianakou et al.,2005).

FNAC has a major important in the management of patients with breast lesions with

an added potential advantage of providing quality prediction of patient outcome,

response of disease to treatment disease and the susceptibility to breast cancer. The

18
Major factors that determines the reliability and efficiency of the method is the quality

of the samples and the experience of the medical staff that performs the aspiration

(Zagorianakou et al.,2005)

As part of the triple assessment of breast lesion FNAC plays its role of importance in

the accurate description of findings and report (Aasmund et al., 2011).

Unlike in FNAC of breast masses which is palpation guided, FNAC with

ultrasonography guidance is more widely used on nonpalpable lesions. Sampling

insufficiency is common with collagenous lesions and in samples aspirated by

pathologist with no experience in the FNAC procedure.

A diagnostic biopsy is recommended when FNAC provides insufficient material.

FNAC is believed to be a secured technique for screening purposes, though it is

moderately less sensitive than core needle biopsy (Aasmund et al., 2011).

The accuracy of FNAC is fully certain and conclusive when experienced

cytopathologists are available to monitor the sufficiency of the aspirated material and

to give recommendation for additional aspirations for ancillary tests when required

(Aasmund et al., 2011).

The international academy of Cytology (IAC) has set up a standard and

comprehensive and approach to Fine Needle aspiration cytology reporting in

categorizing FNAC of breast lesions into C1- Insufficient Material, C2-Benign, C3-

Atypical, C4-Suspicious, C5-Malignant (Andrew et al., 2017).

19
The quality, clarity, and reproducibility of reports across borders, cities, countries are

made effective with Structured reporting and this will assist in patient management,

improve breast health care, and Facilitate further research (Andrew, et al., 2017).

A widely accepted tumor grading system for the histological grading of breast

carcinoma is with the modified Scarf–Bloom–Richardson (SBR) grading system, Thus

the era of neo-adjuvant chemotherapy, requires FNAC reports for prognostication in

the grading of breast carcinoma (Pal et al., 2016).

20
Figure iv: Demonstrating Breast and Thyroid FNAC (Anam, 2017)

21
2.4.2 LIVER FNAC
The use of a finely targeted needle for aspiration helps to obtain a good degree of

sensitivity and specificity as pertaining to the diverse range of the pathologies seen in

liver mostly when correlated with serological tests (Kanica et al., 2018).

An observational, and prospective Research study of 130 adequate aspirates from liver

nodules done at tertiary care hospital within a two year period from selected FNAC

patients presenting with nodular liver mass detected under USG or CT guidance shows

lesions which were categorized into 11 non neoplastic and neoplastic which were

further classified benign (1), primary malignancies 31 Hepatocellular Carcinoma, 4

cholangiocarcinoma 72 Metastasis and 11 undifferentiated malignancies respectively

on cytology (Kanica et al., 2018).

A quick, simple and precise way of identifying the pathology in liver nodules involve

the use of guided FNAC of the liver. It can be a useful modality to triage the patients

and decide further line of management (Kanica et al., 2018).

2.4.3 ORAL AND SALIVARY GLAND LESIONS FNAC

The Salivary gland (SG) are consists of the Parotid gland, the Sublingual gland and the

Submandibular gland. They are majorly found all around the submucosa of the oral

mucosa which includes lips, floor of the mouth, cheek, hard and soft palates and the

lips, tongue, tonsillar areas and oropharynx (Anuj, et al., 2018).

Fine Needle Aspiration cytology is a rapid, reliable and safe diagnostic tool used for

various lesions of the oral cavity and salivary glands (Shubhangi et al., 2018). Salivary
22
gland swelling can result from an inflammatory process, cysts or tumors. Oral and

Oropharyngeal mass lesions are majorly diagnosed by biopsy yet with the use of

FNAC diagnostic testing of lesions is shown to be less invasive, less traumatic and

cheap (Shipra et al., 2018).

Cytological studies have shown that aspiration from salivary gland contains Acinar

cells, ductal epithelial cells and scant fibro vascular stroma with only a low yield of

epithelial cells. The normal structures are seen mainly as acinar cells in well-preserved

cohesive ball-like formations and as ductal cells in monolayer sheet (Anuj, et al.,

2018).

Fine needle aspiration cytology (FNAC) of salivary gland is a common technique with

high sensitivity and specificity (Ruchita et al., 2015). Nonetheless, the interpretation

of FNAC smear of salivary gland lesions pose a major problem to the cytologists.

There are some major resemblance in the cytological features of the various tumors

and other lesions in the head neck region (Ruchita et al., 2015).

Sometimes it could be challenging differentiating benign tumor from malignant one

from the tumor with same cell of origin (Ruchita et al., 2015).

Fine-needle aspiration cytology (FNAC) is an established secured diagnostic tool for

the preoperative assessment of salivary gland lesions. This diagnostic technique is

relatively painless, easy to perform, and not expensive, in addition, it provides helpful

knowledge or information to distinguish between benign and malignant salivary gland

23
tumors which also helps in the management and surgical planning (Ghassan et al.,

2021).

Fine needle aspiration cytology (FNAC) of oral lesions has not been routinely utilized

for diagnosis as a result of the oddity and heterogeneity of lesions, distinctive anatomy

of maxillofacial region, difficulty in aspirating these lesions, and limited experience

(Surbhi et al., 2015).

FNA cytology of the salivary gland is a helpful technique for diagnosis of salivary

gland lesions. Incorrect cytological interpretation results from inadequate cellularity

(Ghassan et al., 2021).

2.4.4 FNAC OF SOFT TISSUE LESIONS AND TUMORS

FNAC is safe, easy, cost efficient primary tool in the diagnosis of neoplastic and non-

neoplastic soft tissue lesions (Anitha et al., 2018).

FNAC plays a very important role in initial diagnosis of soft tissue tumors (Veenu et

al., 2017).

FNAC is also found to be relevant in differentiating neoplastic from non-neoplastic

soft tissue lesions, distinguishing metastatic carcinoma and melanoma in soft tissue

from primary soft tissue tumors and differentiating benign and malignant soft tissue

tumors (Anitha et al., 2018).

The incidence of benign soft tissue tumors is about ten times that of malignant ones.1

Benign deep masses in adults are usually due to intramuscular lipoma. Extremity

masses larger than 5-7 cm and deeper than subcutaneous tissue, favour the diagnosis

24
of a malignant soft tissue tumor. Benign tumors are usually superficial and well

defined or encapsulated masses showing slow growth (Anitha et al., 2018).

Fine Needle aspiration cytology (FNAC) is useful in distinguishing accurately

between benign and malignant soft tissue tumours and sub classify them into general

and clinical relevant cases to initiate treatment. It is a very useful procedure in

preoperative diagnosis of benign and malignant soft tissue tumours with certain

limitations. It is safe, useful procedure of low financial cost.

Fine needle aspiration cytology (FNAC) has been established as one of the first

diagnostic tools in evaluation of soft tissue lesions (Anitha et al., 2018).

FNAC aids in differentiating between neoplastic and non-neoplastic soft tissue lesions,

distinguishing metastatic carcinoma and melanoma in soft tissue from primary soft

tissue tumors and differentiating benign and malignant soft tissue tumors (Anitha et al.,

2018).

Supportive tissue of some different body organs as well as the non-epithelial, extra

skeletal structures are referred to as soft tissues. Examples are the adipose tissue,

fibrous connective tissue, skeletal muscle, blood vessels, and the peripheral nervous

system. Except for the peripheral nerves they mostly have their origin from the

mesoderm.

Soft tissue tumors (STTs) are group of tumors which are highly varied and are

classified based on their similarities to adult tissue on a histogenic basis (Anitha et al.,

2018).

25
The fundamental task of cytopathologists in FNAC of soft tissue lesions is to

determine whether the lesion is neoplastic or reactive and if neoplastic, whether it is

benign or malignant. The occurrence rate of benign soft tissue tumors is about ten

times that of malignant ones (Anitha et al., 2018).

2.5 ROLE OF CELL BLOCK IN FNAC SAMPLE PREPARATION

Fine needle aspiration cytology of superficial lesion or deeper anatomical site is a

rapidly growing technique employed in neoplastic lesion diagnosis.

The usage of cell block forms the solution to the problem encountered when FNA

does not yield sufficient information for precise diagnosis & the risk of false negative

or intermediate diagnosis always exist (Basnet et al., 2012).

The use of scrape cell block technique in addition is essential in cases where repeat

aspiration may not be possible when deep seated organs have been obtained by image

guided techniques. In this technique fixed cellular materials on slides are stained and

carefully removed by scrapping then processing as cell block (Kulkarni et al., 2000).

An indefinite or uncertain diagnostic testing on FNAC is sequel to the presence of

thick tissue fragment and poor spreading even at adequate amount of aspirate

(Kulkarni et al., 2000).

2.5.1 Thyroid Lesion: Cell blocks and smear prepared from a remnant of tissue fluid

acts to supplement each other in order to establish a conclusive diagnosis of thyroid

lesion (Kiran et al., 2021).

26
FNAC is now a prominent method for the cytological analysis of thyroid nodules,

characteristically fast, reliable, safe, minimally invasive and cost effective. Its major

function and purpose is aimed at bringing a clear demarcation between benign and

malignant lesion so as to form a reference point for decision making during treatment.

Cell block and FNA are mostly used as the first test of triage for thyroid lesions. It is

known to play a critical role following decisions of subsequent clinical management of

thyroid nodules (Kiran et al., 2020). At majority of cases after excision, diagnostic

reports remain unchanged, but at other conditions, there are no correlations observed

in reports when benign lesions

are reported on FNAC and cell blocks and are found to be malignant on final

histopathology or when Malignant lesions are reported on FNAC and cell block and

found to be benign on final histopathology (Kiran et al., 2021). Inadequacy of

specimen materials & expertise needed for testing have diminished the various merits

of Fine needle aspiration cytology. Thus Cell block are now commonly used in body

fluid cytology because of its considerable advantage of maximum accuracy in

diagnosis (Vaishali et al., 2020).

Conventional smear using FNAC has remain a preferred method for thyroid lesion

diagnosis, though with minimal sample adequacy, and inter-individual variations.

Although liquid based cytology techniques is a modified technique developed for the

purpose of overcoming limitations of conventional smear, yet its clinical utility and

accuracy over conventional smear are controversial (Yosep et al., 2017).

27
2.5.2 Tumor: FNAC guided with ultrasound procedure is a preoperative diagnostic

procedure in various deep seated neoplastic and non-neoplastic mass lesion. Cell block

prepared for remnant FNA material aid in improved morphologic assessment &

contribute to develop a more definitive cytopathology diagnosis (Sumana et al., 2015).

2.5.3 Oral cytopathology: Cell block and fine needle aspiration can be used for oral

cytopathology testing. Conventional FNA smear gives insufficient diagnostic material

while in cell block techniques, provides more tissue resulting in enhanced accuracy of

diagnosis. Research shows that modified cell block gives an excellent cytopathology

features than fine needle aspiration cytology (Sale et al., 2020).

2.5.4 Breast Lesions: False positive and false negative diagnostic results pose a

limitation in the accuracy of diagnosis of Breast malignancies with Fine needle

aspiration (Fred et al., 2020). However, Cell block preparation have been

recommended by pathology researchers to supplement the diagnostic accuracy of

FNAC (Fred et al., 2020).

2.6.0 SMEAR SAMPLING TECHNIQUES IN FNAC

Artifacts that arise from the smear sampling techniques have not been adequately

described, though the quality of cytological preparations with the use of LBC have

been well documented (Mydakos et al., 2009)

28
Liquid-based cytology is a common technique used in the cytology lab for basic

sample preparation. This technique was first tried on Pap smear after being introduced

and then finally gained approval from the Food and Drug Association (FDA) in

1996(Koss et al., 2005).

Liquid-based cytology is performed not only for gynecological cytology but also for

non-gynecological cytology such as the conventional fine needle aspiration cytology

(FNAC), guided Fine Needle aspiration cytology and some other fluid cytology.

The thin-prep (TP) smears are smears that have the characteristic of being properly

preserved or kept in its original state and evenly dispersed with no products of

unknown cells elements.

The only major problem found on the liquid based cytology smears are clusters of

small-sized cell with many single cells than sheets. The cells are commonly smaller,

with a reduced chromatin structure. Though the nucleoli are more prominent, the

intranuclear inclusion is difficult to visualize, and yet there is a minimum level of

visible extracellular elements. It also characterized by a scanty number of small red

blood cells, myoepithelial cells and some mononuclear cells with the background

matrix being modified (Kalpalata et al., 2015).

Liquid-based cytology specimen is obtained commonly with a small brush, similar to

the conventional smear method, but in contrast the sample is being transferred into a

vial of preservative liquid. The sample is subsequently being treated to remove other

products such as mucus before a layer of the cell sample is placed on a slide. The

29
method gives room for a more quality result. Liquid-based cytology (LBC) is a good

technique for examining most cytological preparations with no limitation to fine

needle aspiration as a result of the even distribution of cells, low level of artefacts such

as blood and mucus, and a well preserved nuclear and cytoplasmic details (Kalpalata

et al., 2015).

Liquid based cytology in Fine Needle aspiration smears can carried out on aspirates

from various body organs such as the breast, bone, salivary gland, thyroid, lymph

nodes, and some other special fine needle specimen (Khan et al., 2012).

Conventional smear technique is a well-known diagnostic option for thyroid lesion in

spite of insufficient sample availability. Liquid based cytology is a technique

developed with the aim to meet up the limitation constraint.

Primarily materials aspirated with a fine needle is expelled directly onto the

microscope slide for a smear preparation. Preparing a conventional smear entails

expelling materials aspirated with a fine needle onto a microscope slide (Neeraja et al,

2015).

Liquid based Cytology characteristically possess a good nuclear and cytoplasmic

details with no ambiguous background materials (Priya, 2016).

30
2.7 LIMITATIONS OF CONVENTIONAL FINE NEEDLE ASPIRATION
SMEAR
Parts of the limitation of a conventional FNA smear is the insufficient availability of

materials to facilitate other diagnostic testing such as immunocytochemistry (Shehnaz

et al., 2012).

31
Figure v: Thin Prep Smear (Anam, 2017).

32
 CHAPTER THREE
3.0 MATERIALS AND METHOD

3.1 MATERIALS
Cytological Materials such as slides, cover slip, cellular base, Preservative vials,
cleaning solution and others were used for the smear sampling techniques while
Tissue Processor, embedding machine, Microtome, Tissue cassette, Histogel and other
histological materials were used for the Cell block technique.

3.1.1 Histogel
The thermo Scientific Richard-Allan Histogel was used for this project. It is an
aqueous gel composition useful in processing histological and cytological specimen. It
is usually refrigerated when not in use and kept out of direct light.

3.1.2 Liquid based cytology kit

The liquid based cytology kit enhances diagnostic accuracy and gives a better
preservation of cells. It ensures an efficient collection because it comes as a collective
kit where you can collect and fix samples directly on to the slide.

3.2 METHODOLOGY

3.2.1 Sample Collection for Conventional Staining

Samples were collected using the necessary equipment from the appropriate
anatomical sites and sent to the laboratory immediately. Smears were prepared
according to the requirement of the stain to be used.

50 samples were observed macroscopically, documented, labelled and suspended. 1

slide was made for Pap staining to demonstrate cells of epithelial origin (PAP), 1 slide
for H/E staining to demonstrate general cellular structure, and 1 slide for (RRS) to
demonstrate cells of non-epithelial origin making a total of 3 slides prepared for each

33
samples and a total of 150 slides obtained from the 50 samples meant for conventional
staining.

3.2.2 Sample Preparation for Liquid based Cytology Staining

In LBC samples were prepared with the following method

1. 2 drops of sample were placed in a clean test tube

2. 2ml of cleaning solution was then added to the test tube and spun using a centrifuge
at 4000 rpm for 10 minutes.

3. The supernatant was decanted and the residue was left in the test tube after
spinning.

4. 1 ml of the cellular base was then added to the residue and the mixture was agitated
with a vortex for proper mixing for few seconds.

5. 50 micro-liters of the mixture was then taken using an automatic pipette and a
circular smear was made on a slide and left to dry on a hot plate.

6. After drying, the slides were stained using Papinicoloau staining technique,
Hematoxylin & Eosin staining technique and Rapid Romanowsky staining technique.

The above technique was used for 50 samples and 2 slides were made for Pap stain 1
slide for RRS stain and 1 slide for H/E stain making a total of 4 slides prepared for
each of the samples and a total of 200 slides obtained from the 50 samples meant for
cytological evaluation.

3.2.3 Rapid Romanowsky Stain

Principle

The Proprietary brand of a romanowsky stain are defined as being the black precipitate
formed from the addition of aqueous solutions of methylene blue and eosin, dissolved
in Methanol. The variants of the Romanowsky group differ in the degree of oxidation
(polychroming) of the methylene blue stain prior to the precipitation. Romanowsky
34
stains are universally employed for staining blood films and are generally very
satisfactory. The main components of a Romanowsky stain are:

1. A cationic or basic dye such as Azure B, which binds to anionic sites and gives a
blue grey color to nucleic acids (DNA or RNA), nucleoproteins, granules of basophils
and weakly to granules of neutrophils.

2. An anionic or acidic dye such as Eosin Y, which binds to cationic sites on proteins
and gives an orange-red colour to haemoglobin and eosinophil granules.

Procedure:

1. Smears were fixed in 70% Alcohol for few minutes

2. Smears were stained in Solution 1 (Primary Stain) for 2 minutes.

3. Smears were stained in Solution 2 (Counter Stain) for 5 minutes.

4. Smears were rinsed in distilled water.

5. Smears were taken to Absolute Alcohol (1) and (2) for 5 minutes each.

6. Slide were Cleared in Xylene and mounted with DPX.

3.2.4 Hematoxylin and Eosin staining

Principle: The Hematoxylin (basic dye) will stain the acidic property of the cell
(nucleus), while the acidic dye (eosin) will stain the basic property of the cell
(cytoplasm).

Procedure:

1. The slides were rinsed in Absolute Alcohol for 30 seconds

2. The slides were taken to 96%Alcohol for 30 seconds

3. The Slides were taken to 70% Alcohol for 30 seconds

4. The slides were taken to 50% Alcohol for 30 seconds

35
5. The Slides were rinsed in Distilled water for 30 seconds

6. The slides were Stained in Harris Hematoxylin solution for 5 minutes.

7. The slides were rinsed in water

8. The slides were blued in tap water for 5 minutes.

10. The slides were counterstained with eosin briefly

11. The slide were dehydrated in Ascending grade of Alcohol 70%, 90% and 96% of
Alcohol and Absolute Alcohol for 30 seconds each.

12. The slides were cleared with xylene and mounted with DPX.

3.2.5 Papinicolaou Staining

Principle: This is the most widely used staining procedure for cytological specimen. In
the first step, the nuclei are stained by a Hematoxylin solution. Nuclei are stained blue,
dark violet to black. The second staining step is cytoplasmic staining by Orange
staining solution, especially for demonstration of mature and keratinized cells. The
target t structures are stained orange in different intensities. In the third staining step,
the so-called polychromatic solution is used (Azure Eosin). The polychromatic
solution is used for demonstration of differentiation of squamous cells.

Procedure:

1. Fixed smears were passed through descending grades of Alcohol (96%, 70%, and
50%) for 30 seconds each.

2.Smears was rinsed in distilled water for 10 seconds

3. Smears was stained in Harris Hematoxylin solution for 5 minutes.

4. Smears were rinsed in water and blued in tap water for 5 minutes

5.Smears were rinsed in 2 changes of 96% Alcohol 30 seconds each.

6.Smears were stained in Papinicolaou stain 0G6 solution for 3 minutes

36
7. Smear was rinsed in 2 changes of 96% Alcohol for 30 seconds.

9. Smears were stained in Papanicolau solution EA50 for 3 min

11. Smears were dehydrated with Absolute Alcohol (1) and (2) for 30 seconds each.

19. Smears were dehydrated with equal parts of Absolute alcohol and Xylene for 5
minutes.

20 Smears were cleared with xylene for 5 minutes and mounted with DPX.

3.2.6 Cell Block Processsing

Left over of samples collected in vials after preparation of smears were used for cell
block preparation with the following method

1. The Histogel which is solid at room temperature was liquefied for use by heating
with the aid of a hot air oven.

2.4 to 6 drops of liquefied Histogel was pipetted into the cell pellets at the bottom of
the centrifuge tube. The Mixture was vortexed for few seconds to adequately and
thoroughly mix the cells and the histogel together. The histogel was allowed to
solidify with the cell pellets by cooling to near room temperature (<20 0c). with a
refrigerator.

3. The solidified cell pellets was removed from the tubes and was wrapped
appropriately with a filter paper, placed in a Tissue cassette and taken to the
Automatic tissue processor for processing.

4. Cell pellets were then embedded after processing and sections were cut at 3
microns.

5. Ribbons of section was spread out with 20% alcohol, floated in a warm water bath,
and desirable sections of cell pellets were picked on a slide

37
6. The slides were dried with a hot plates and were arranged in staining racks for
staining.

The above technique was used for 50 samples and 1 slides were made for Pap stain 1
slide for RRS stain and 1 slide for H/E stain making a total of 3 slides prepared for
each of the samples and a total of 150 slides obtained from the 50 samples meant for
cytological evaluation.

3.2.7 Staining Procedure for Cell Block sections

3.2.7.1 Rapid Romanowsky staining

1. Slides were dewaxed in 3 changes of xylene for 5 minutes each.

2. Slide were passed through descending grades of Alcohol. Absolute Alcohol, 96%
Alcohol, 70% Alcohol for 5 minutes each.

3. Slides were stained in Solution 1 (Primary Stain) for 2 minutes

4. Slide were stained in Solution2 (Counter Stain) for 5 minutes.

5. Slide were Rinse in Water

6.Sliides were dehydrated in Absolute Alcohol (1) and (2) for 5 minutes each.

7. Slides were cleared in Xylene

8. Slides were mounted with DPX.

3.2.7.2 Hematoxylin and Eosin staining

Principle: The Hematoxylin (basic dye) will stain the acidic property of the cell
(nucleus), while the acidic dye (eosin) will stain the basic property of the cell
(cytoplasm).

38
Procedure:

1. The slides were dewaxed in 3 changes of Xylene for 5 minutes each.

2. The slide were passed through descending Grades of Alcohol Absolute Alcohol,
96% Alcohol, and 70% Alcohol for 5 minutes each.

3. The Slide were rinsed in water

4. The slides were stained Gills Hematoxylin for 10 minutes

5. The Slides were rinsed in water

6. The slides were differentiated in 1% Acid Alcohol for few seconds

7. The slides were rinsed in tap water and Blued in running tap water.

8. The slides were counter-stained in eosin for 1 minutes.

9. The slides were rinsed in water

10. The slide were dehydrated in Ascending grade of Alcohol 70%, 90% and 96% of
Alcohol for 10 dips each.

11. The slide were dehydrated in Absolute alcohol for 5 minutes and then cleared with
3 changes of xylene for 5 minutes each.

12. The slides were Mounted with DPX.

3.2.7.3 Papinicolaou Staining

Principle: This is the most widely used staining procedure for cytological specimen. In
the first step, the nuclei are stained by a Hematoxylin solution. Nuclei are satined blue,
dark violet to black. The second staining step is cytoplasmic staining by Orange
staining solution, especially for demonstration of mature and keratinized cells. The
target structures are stained orange in different intensities. In the third staining step,

39
the so-called polychromatic solution is used (Azure Eosin). The polychromatic
solution is used for demonstration of differentiation of squamous cells.

Procedure:

1. Sections were dewaxed in 3 changes of xylene for 5 minutes each

2. Slides were passed through descending grades of Alcohol (Absolute Alcohol 96%
Alcohol and 70% Alcohol for 5 minutes each.

2.Slides were rinsed in water

3. Slides were stained in Harris Hematoxylin solution for 10 minutes.

4. Slides were rinsed in water and blued in tap water for 5 minutes

5.Slides were rinsed in 2 changes of 96% Alcohol 30 seconds each.

6.Slides were stained in Papinicolaou stain 0G6 solution for 3 minutes

7. Slides was rinsed in 2 changes of 96% Alcohol for 30 seconds.

9. Slides were stained in Papanicolau solution EA50 for 3 min

10. Slides were dehydrated in 2 changes of 96% Alcohol.

11. Slides were dehydrated with Absolute Alcohol (1) and (2) for 30 seconds each.

12. Slides were cleared with xylene for 5 minutes and mounted with DPX.

3.3. ETHICAL APPROVAL

Ethical Approval was sought from the ministry of health with a Ref Number of
HA.821/03

40
3.4 LOCATION OF STUDY
This study was carried out in the University of Benin Teaching Hospital, in Ugbowo
Benin City. The Hospital is situated in Ovia North, Edo state Nigeria. Its geographical
coordinates are 6024 0 North, 50 361 0" East. (Osaro et al., 2010).

41
 CHAPTER FOUR
4.0 RESULTS
A total of 500 Slides were obtained from the 50 samples meant for cytological
evaluation. Some representative samples of the stained slides were viewed with the
microscope and photomicrographs of slides were obtained. Results of cell block slides
and that of the smear sampling technique were both compared for preservation of
cellular morphology, diagnostic accuracy, microscopic appearance and cost efficiency.

PHOTOMICROGRAPHS OF RANDOM SAMPLES OF SELECTED SLIDES

X 400 MAGNIFICATION CELL BLOCK PREPARATION (ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Ascitic fluid cell block reveals adequate clusters and morulesS of atypical epithelial
cells with hyperchromatic nuclei and irregular nuclear membrane (long arrow). The
background appears haemorrhagic (short arrow) and suggestive of a malignant smear.

42
X 400 MAGNIFICATION CONVENTIONAL CYTOCENTRIFUGATION
METHOD PREPARATION (ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Ascitic fluid conventional smear reveals not so prominent clusters and morules of
atypical epithelial cells. There is loss of distinct hyperchromatic nuclei and irregular
nuclear membrane (long arrow) when compared to the liquid based cytology and cell
block method. The background appears densely haemorrhagic (short arrow) and smear
diagnosis inconclusive.

43
X 400 MAGNIFICATION LIQUID BASED CYTOLOGY PREPARATION
(ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

PLATE 1:
Ascitic fluid Liquid based cytology smear reveals prominent clusters and
morules of atypical epithelial cells with loss of distinct hyperchromatic
nuclei and irregular nuclear membrane (long arrow) as seen in cell block
method. The background appears clear (short arrow) and highly
suggestive of a malignant smear.

44
X 400 MAGNIFICATION CELL BLOCK PREPARATION (ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Ascitic fluid cell block reveals adequate predominantly signet ring cells with
pleomorphic eccentrically placed hyperchromatic nuclei (long arrow). The background
appears haemorrhagic with scanty lymphocytes (short arrow) and suggestive of a
malignant smear probably a mucin producing adenocarcinoma

X 400 MAGNIFICATION CONVENTIONAL CYTOCENTRIFUGATION

METHOD PREPARATION (ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Ascitic fluid cytocentrifugation method reveals a busy smear with scanty signet ring
cells with pleomorphic eccentrically placed hyperchromatic nuclei (long arrow)
45
occluded in a background that appears haemorrhagic with scanty lymphocytes (short
arrow) and suggestive of a malignant smear probably a mucin producing
adenocarcinoma

X 400 MAGNIFICATION LIQUID BASED CYTOLOGY PREPARATION

(ASCITIC FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

PLATE 2: Ascitic fluid liquid based cytology method smear reveals adequate signet
ring cells with pleomorphic eccentrically placed hyperchromatic nuclei
(long arrow) in a clear background that appears haemorrhagic with scanty
lymphocytes (short arrow) and suggestive of a malignant smear probably
a mucin producing adenocarcinoma

46
X 400 MAGNIFICATION CELL BLOCK PREPARATION (PLEURAL FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Pleural fluid cell block reveals scanty epithelial cells with pyknotic nuclei (long
arrow). The background appears clear with scanty lymphocytes and inflammatory
cells (short arrow) and suggestive of a benign smear.

X 400 MAGNIFICATION CONVENTIONAL CYTOCENTRIFUGATION

METHOD PREPARATION (PLEURAL FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

47
Pleural fluid cytocentrifugation method reveals not so prominent epithelial cells with
pyknotic nuclei (long arrow). The background appears haemorrhagic with scanty
lymphocytes and inflammatory cells (short arrow) and suggestive of a benign smear.

X 400 MAGNIFICATION LIQUID BASED CYTOLOGY PREPARATION

(PLEURAL FLUID)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

PLATE 3: Pleural fluid liquid based cytology method reveals adequate

distinct epithelial cells with pyknotic nuclei (long arrow). The
background appears neat and clear with scanty lymphocytes and
inflammatory cells (short arrow) and suggestive of a benign smear.

48
X 400 MAGNIFICATION CELL BLOCK PREPARATION (BREAST CYST)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Breast cyst cell block reveals predominantly foamy macrophages (long arrow), with
scanty lymphocytes and inflammatory cells mixed with plasmacytic cells in a
background of haemorrhage (short arrow) and suggestive of a benign smear.

49
X 400 MAGNIFICATION CONVENTIONAL CYTOCENTRIFUGATION
METHOD PREPARATION (BREAST CYST)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

Breast cyst conventional cytocentrifugation method reveals predominantly foamy

macrophages (long arrow), with scanty lymphocytes and inflammatory cells mixed
with plasmacytic cells in a background of haemorrhage (short arrow) and suggestive
of a benign smear.

50
X 400 MAGNIFICATION LIQUID BASED CYTOLOGY PREPARATION
(BREAST CYST)

Papanicoula Stain H&E Staining Rapid Romanowsky Stain

PLATE 4; Breast cyst liquid based cytology reveals predominantly foamy

macrophages (long arrow), with scanty clusters of cytological normal
polygonal to spindle shaped cells and mild mixed inflammatory infiltrates
of neutrophils and lymphoplasmacytic cells in a clear neat background
(short arrow) and suggestive of a benign smear.

51
 CHAPTER FIVE
5.0 DISCUSSION SUMMARY AND CONCLUSION

5.1 DISCUSSION
LBC has been found to have a peculiar major advantage in lowering the rate of
unsatisfactory smears (Aminisani et al., 2013).
The smear sampling technique with liquid based cytology allows cells to be suspended
in a fluid medium and dispersed in a monolayer for a better morphologic examination
(Prajikta et al., 2014).
Findings from this study shows that the smear sampling technique with liquid based
cytology shows an advantage due to cleaner background and reproducibility, better
cellular morphology and ability to gain sample for ancillary technique.
Findings from this studies also shows that certain sample preparation demonstrated a
clear morphology in cell block as compared to liquid based cytology. The
pathophysiological mechanism behind the formation of ascitic fluid determines the
quality and types of cytological cells examined for diagnosis. Smear cytology is of
Profound importance when there are clinical cases of suspected malignancy (Rhada et
al., 2019). A common problem observed with conventional smear cytology is the
increase false negative rate which could be as a result of some underlying mechanism
by which malignant tumours cause ascites and still remains negative on cytology.
According to Motherby et al. (1999) A high false negative rate could result from
errors in diagnosis by the cytologist, Errors in Laboratory methods or an underlying
result of effusion not connected to malignancy or even due to sub optimal sampling
error (Rhada et al.,2019). Findings from this study proves that the limitations of
conventional smears such as overspreading of cells, profusion of inflammatory cells,
inadequacy of representative cells or cell loss can be improved on through the Cell
block technique or Liquid based cytology technique. The cell block techniques give a
defined morphological and architectural characteristic which provides clarity in
52
differentiating malignant from non-malignant cells during microscopic examination
(Theerada et al., 2017). Findings from this study as observed in the Photomicrographs
of ascitic, pleural and breast cyst aspirate samples shown in the result above indicates
that there are characteristic differences in morphological demonstrations of cellular
component with respect to the techniques and the stains employed for demonstration.
From the study of Godwin et al it was shown that the Liquid based cytology technique
with Needle Hub on Breast Malignancy cases has a pronounced importance in
cytological detection. Findings from this study also shows that Cell block facilitate the
easy identification of the histological pattern of breast cyst aspirate compared to the
smear sampling techniques. Cystic fluid aspirate is only routinely demonstrated by
cytology techniques when they are turbid, cloudy or with blood stains (Paulo et al.,
2011). Cytology smears prepared from this fluid are commonly observable with
inflammatory cells mixed with other macrophages as a confirmatory characteristics of
the cystic nature of the lesion (Paulo et al., 2011). Specimen or smear adequacy is
determined by the nature of the lesion, experience of the pathologist and techniques
employed in obtaining the specimen. Certain factors such as degenerated apocrine
cells, necrosis, and epithelial hyperplasia are usually discovered when examining
difficult smears. Some other causes of a false negative diagnosis include; non palpable
breast lesion with small tumor size. All this are factors considered for interpreting the
diagnosis of a breast FNAC as benign (Paulo et al., 2011).
From economic and financial consideration, findings from this study shows that the
Cell block techniques is more expensive for consideration when it is limited to smear
assessment or meant for only cytological diagnosis and not required any for a further
ancillary studies. This finding is confirmed by the principle that decisions making on
options of diagnostic technique is considered based on the procedures required, price,
and availability of needed materials (Alves et al., 2004). The manual method of liquid
based sampling technique is less expensive because it doesn’t require sophisticated
devices for preparation (Maskem et al., 2001). The liquid based cytology technique

53
also possess the capacity of providing residual samples for DNA testing (Aminisani et
al., 2013).
Liquid-based cytology (LBC) is a good technique for examining most cytological
preparations with no limitation to fine needle aspiration as a result of the even
distribution of cells, low level of artefacts such as blood and mucus, and a well
preserved nuclear and cytoplasmic details (Kalpalata et al., 2015). One of the major
goals in demonstration of FNAC samples with the liquid based cytology technique and
Smear technique is to differentiate benign from malignant lesions. However, the
presence of only small amount of sample for use and morphological overlap between
both lesions are some limitations to this goals (Paulo et al., 2011).

Liquid base cytology in Fine Needle aspiration smears can carried out on aspirates
from various body organs such as the breast, bone, salivary gland, thyroid, lymph
nodes, and some other special fine needle specimen (Khan et al., 2012).

The thin-prep (TP) conventional smears are smears that have the characteristic of
being properly preserved or kept in its original state and evenly dispersed with no
products of unknown cells elements. The only problem found on the liquid base
cytology smears are clusters of small-sized cell with many single cells than sheets.
The cells are commonly smaller, with a reduced chromatin structure. Though the
nucleoli are more prominent, the intranuclear inclusion is difficult to visualize, and yet
there is a minimum level of visible extracellular elements. It also characterized by a
scanty number of small red blood cells, myoepithelial cells and some mononuclear
cells with the background matrix being modified (Kalpalata et al., 2015). Cell block
are essential in fine-needle aspiration biopsy (FNAB) and there are various benefits in
a well-prepared, diagnostic CB. The Routine hematoxylin and eosin (H & E) stained
sections of cell block with high quality is essential for a smear where both the detailed
cytological and architectural structure is desired to give room for the cytomorphologic
findings on it (Khan et al., 2012).

54
A quality cell block is indispensable for cytochemical staining as well as
immunohistochemical staining and other molecular diagnostic studies like polymerase
chain reaction (PCR), and fluorescence in situ hybridization (FIS) (Symanns et al.,
2003).

5.2 SUMMARY
A major unique relevance of cell block compared to other smear sampling techniques
is the capacity of providing several sections for a clear and efficient microscopic
demonstration with added advantage of storing the blocks for future ancillary
techniques. The demerits encountered with cell block are delayed time of diagnosis
and in some cases the risk of losing materials during the Processing.
It is essential to combine the Cell block techniques and the smear sampling technique
when a rich or quality diagnosis is desired. Fine Needle aspiration cytology is mostly
utilized for diagnosis in pathology because of its characteristic accuracy, sensitivity
and specificity. The various similar advantages encountered with the uses of the
Liquid based methods and cell block method of analysis include: the complete capture
of all cellular materials, Specimen randomization, improved preservation of Cellular
sample, lowered level of obscuration. False negatives or false positive diagnosis may
not always result from using a conventional smear but may also be due to a number of
some other varying causes which could be attributed to the nature of the sample, poor
localization, and sub optimal sampling techniques or in other cases may be due to
interpretation errors with respect to false positive cases. From the economic
consideration for smear assessment the conventional method forms the cheapest
method for smear analysis while the liquid based cytology technique is less high
compared to the cell block method.

55
5.3 CONCLUSION
For cytological diagnosis Liquid base cytology techniques renders a huge importance
not only in inflammatory or hemorrhagic samples but also in providing better
morphological appearances, even dispersion or spread of smears, and offers an good
turnaround time for diagnostic result unlike the cell block technique.
The cell block technique plays a more useful role in Preserving of cytological
specimen for over a long period of time and this is the best choice of option when
specimen will be required for future studies. There is a profound improvement in
diagnostic cytology of FNAC and other Non-gynecological samples as a result of the
simultaneous utilization of smear sampling techniques and Cell block.
Microscopic findings of stained cell block sections are more effective in
demonstrating positivity at a higher level of the cases in the conditions of malignancy.
Cell blocks give a higher level of clarified diagnosis when classifying suspicious
lesions.

5.4 RECOMMENDATION
Further studies should be done with Cell block techniques as a medium to improve
diagnostics knowledge of ancillary techniques and studies on Immunohistochemistry,
Cellular markers and Insitu Hybridization.

56
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