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Invited Review Article

Indian Academy of Cytologists Guidelines for Collection,

Preparation, Interpretation, and Reporting of Serous Effusion
Fluid Samples
Effusion Guidelines Committee of IAC
Guidelines drafting and finalization committee

Radhika Srinivasan, Bharat Rekhi, Arvind Rajwanshi, Saleem Pathuthara, Sandeep Mathur, Deepali Jain, Nalini Gupta, Upasana Gautam, Naresh Rai,
Vijay Shrawan Nijhawan, Venkat Iyer, Pranab Dey, Prabal Deb, Dev Prasoon
Indian Academy of Cytologists, India

Abstract
Cytological examination plays an important role in the initial work‑up of the serous cavity effusion fluids to find out the possible etiology as
benign or malignant. Among malignant effusions, cytology is helpful in determining the exact type, site, and stage of the tumor. However,
for reporting effusion cytology specimens, there is no consistent and reproducible reporting system. Aims: The aim of these guidelines is
to provide a standardized format for effusion cytopathology right from sample receipt to its ultimate report sign‑out for implementation in
all cytopathology laboratories. The Indian Academy of Cytologists in consultation with experts across the country has prepared guidelines
pertaining to collection, preparation, and diagnostic categories of effusion specimens to reduce reporting variability. The guidelines are made
keeping in mind the different areas of practices in India, especially low‑ and medium‑resource settings. The guidelines are broadly divided
into essential, optimal, and optional categories for best usage and appropriate allocation of the precious specimens. In referral centers or
well‑established setups, essential ancillary techniques can be done for accurate and final diagnosis. By adhering to and implementing these
uniform guidelines, it is hoped that clinical patient care and management in India will improve and be of uniformly good quality by enabling
and facilitating good laboratory practices.

Keywords: Categories, cell block, cytopreparation, effusion cytology, guidelines, immunocytochemistry, immunohistochemistry, Indian
Academy of Cytologists, reporting

Introduction To achieve uniformity in this process across our country, the

Serous effusion indicates accumulation of excess fluid in the
body cavities, namely, pleural, pericardial, and peritoneal, the
latter also referred to as ascites. Effusion invariably indicates an
underlying pathology and constitutes an important diagnostic
sample in clinical practice, including oncology.[1]
Specimen from various anatomic sites can be evaluated
by cytology. The techniques for collection, transportation,
and preparation of specimen are of prime importance, as
an adequate, well‑prepared, well‑stained smear helps in the
ultimate goal of an accurate cytopathological diagnosis.[2]
The methods of collection and processing of effusion specimen
for cytological diagnosis vary from laboratory to laboratory.
Access this article online
Quick Response Code:
Website:
www.jcytol.org
Indian Academy of Cytologists  (IAC) has developed these
guidelines in consultation with experts across the country for
implementation as a standard format in our country for all
laboratories providing cytopathology services.
The IAC guidelines are broadly divided into three main
categories as follows:
i. ESSENTIAL: these are absolute and non‑negotiable
recommendations, and if a laboratory cannot achieve
Address for correspondence: Dr. Radhika Srinivasan,
Secretary, Indian Academy of Cytologists.
E-mail:secty.iac@gmail.com
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix,
tweak, and build upon the work non-commercially, as long as appropriate credit is given and
the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

How to cite this article: Effusion Guidelines Committee of IAC. Indian

DOI: academy of cytologists guidelines for collection, preparation, interpretation,
10.4103/JOC.JOC_157_19 and reporting of serous effusion fluid samples. J Cytol 2020;37:1-11.
Submission: 02-12-2019; Revision: 04-12-2019; Acceptance: 05-12-2019; Publication: 23-12-2019

© 2019 Journal of Cytology | Indian Academy of Cytologists | Published by Wolters Kluwer - Medknow 1

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IAC effusion fluid cytology guidelines
this, then the sample may be referred to a center which is
equipped to fulfil these criteria.
ii. OPTIMAL: this is achievable in any good laboratory and
should be part of the protocol and can be made mandatory
for accreditation.
iii. OPTIONAL: this recommendation is resource‑dependent
and is left to the choice of the individual laboratory.
The aim of preparing IAC guidelines is to achieve good clinical
practice across all laboratories offering cytopathology services
for accurate diagnosis in the interest of patient management.
It is also envisaged that there is a general uniformity in the
recommendations made by the laboratories in the same
situation.
Sample Collection and Transportation
Requisition form  fluid, pericardial fluid, peritoneal washing, and rarely pleural
Essential
Clinical details
The sample should accompany a requisition form with the
following necessary details [Table 1]:
• Name, age, gender, hospital registration number
• Clinical diagnosis, including indications for doing the
procedure
• Date and time of collection
• Date and time of receiving sample in the laboratory
• Temperature conditions in case the sample is
stored – refrigerated or room temperature
• Clinical symptoms
• Imaging finding
• Endoscopic findings, if any
• Previous diagnoses and pertinent treatment history
• Anticoagulant used or not, including type.
Optional
• Tobacco and alcohol habits
• Occupational history
Table 1: Sample requisition form
Name: ABC………… Age/Sex: 50/female
Hospital registration number: 123456
Procedure: Left paracentesis
Sample: Ascitic fluid Amount sent: 40mL
Date and time of collection: Nov 30, 2019. 10AM
Anticoagulant used or not, including type: Heparin
Clinical symptoms/history: Weight loss and abdominal distension
Clinical diagnosis: Tuberculosis
Imaging findings: Left Adnexal mass
Endoscopic findings, if any:
Any other relevant investigations: CA‑125 raised
Relevant past history, if any: None
Investigation required: Evaluation for Malignant cells
2 Journal of Cytology  ¦  Volume 37  ¦  Issue 1  ¦  January-March 2020
Note on effusion sample collection guidelines
Fluid collection is performed by the treating clinicians. The
procedure consists of inserting a wide‑bore needle (under local
anesthesia) through the body wall into the fluid‑containing
cavity. Pleural fluid is removed by thoracocentesis,
peritoneal fluid by paracentesis, and pericardial fluid by
pericardiocentesis.
Peritoneal washing samples may be obtained by instilling
normal saline solution into the various recesses of peritoneal
cavity and then withdrawing the fluid. This is done in patients
undergoing abdominopelvic surgical exploration to detect
peritoneal dissemination of cancer cells.
Collection of fluid sample
Types of samples
The various samples include pleural fluid, peritoneal/ascitic
washing.
Essential
The fluid is collected in a sterile/nonsterile clean dry container
with proper labeling and identification which includes the name
of the patient and hospital registration number.
The laboratory has a right to reject any improperly labeled
samples and those in dirty, cracked, and broken containers
without proper lid.
The laboratory should have a rejection policy for any
improperly labeled samples and those in dirty, cracked, and
broken containers without proper lid.
Recommended volume for cytological evaluation
At least 20–30  mL is optimal. There is no minimum or
maximum limit.
If cell block  (CB) preparation is required, then at least
30–50 mL is optimal. Low cellularity specimen would need
larger volume of fluid.[3,4]
Transportation
Essential
The fluid sample must be transported to the laboratory as
soon as possible as freshly tapped samples are preferred for
cytological examination. Up to 2 hours of transportation time
is achievable in most centers.
If for unavoidable reason processing cannot be done immediately
or within 2 hours, the fluid should be refrigerated at 4°C and
transported on ice/cool box, to arrest degeneration of cells.
Specimen should not be allowed to freeze. Routine cytological
evaluation is possible up to 48 hours under these conditions, but
cell morphology gets compromised after 24 hours.
Anticoagulation – Optimal
In samples with high‑protein content such as exudates and
those which are blood‑tinged or hemorrhagic, addition of an
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IAC effusion fluid cytology guidelines

anticoagulant prevents clotting of sample and can provide • Cover slips

accurate cell counts if required and also achieves optimal smear • A wide‑mouth specimen bottle with fixative
quality. • Normal saline
• Glass marking diamond pencil
The choice of anticoagulants are as follows:
• Mayer’s egg albumin.
i. Heparin easily available in OPDs/wards – 3 units/mL (can
be titrated depending on the strength of heparin). Usually, Optimal
the syringe used for paracentesis or collection container
Biosafety cabinets
is rinsed in heparin.
ii. Citrate Cytocentrifuge machine, funnels, clips, and blotting cards
iii. EDTA – ethylenediaminetetraacetic acid
Applicator sticks with thin tightly wound cotton
iv. Ammonium oxalate – 1% to be added in a ratio of nine
parts fluid to one part anticoagulant. This is a cheap option. Positively charged slides.

Note: NO fixative should be added to the fluid. Formalin

Processing
Essential
prevents cells from adhering to the slide and interferes with
the quality of Papanicoloau staining. Alcohol should not be Technician handling the fluid specimen should wear adequate
added as it causes precipitation of proteins and thus interferes personal protective gear.
with the adherence of the cells to slide.
Centrifuge all samples with a desktop laboratory centrifuge.

Cytopreparation of Effusion Samples In case a fibrin clot is formed, it should be thoroughly smashed
with an applicator stick. In case a large clot remains, it may
Receipt of sample in cytopathology laboratory be processed as CB.
Essential
A representative volume of the fluid (10–15 mL) should be
Upon receipt of the sample, the laboratory generates its centrifuged using capped plastic tubes, 2000 rpm for 10 min.
accession number as per individual laboratory/hospital policy
and the sample is accepted for processing after checking of Subsequently, most of the supernatant should be gently
patient identifiers on the request form and on the sample label. decanted/removed. Care should be taken not to disrupt the
intact sediment while decanting, as it may require repeat
Optimal
centrifugation. From the sediment, smears are made.
Barcoding of sample containers is a common practice.
• A small volume of the sediment using an applicator/
Gross evaluation disposable Pasteur pipette should be placed onto a pre‑labeled
Essential albuminized slide. Thereafter, the sediment should be
spread gently using the flat surface of another glass slide or
Gross or macroscopic evaluation must be performed by the
optimally by rolling the cotton tipped applicator stick gently
laboratory technician/cytotechnician and the following points
in a rotatory motion on the slide, so as to make a thin evenly
are to be noted:
spread smear. Note that the cotton tipped applicator should be
i. Quantity – mL/L moistened by the last drop of fluid at the time of decantation
ii. Color  –  clear/straw‑colored/yellow/brown/red/chylous/ before picking up the sediment for good preparation.
purulent/hemorrhagic/other (please specify) • A minimum of two smears should be prepared from each
iii. Consistency: serous/mucoid/gelatinous/thick copious/ sample – one is air‑dried and another is wet‑fixed/fixed
tar‑like/other immediately in a coplin jar/jar containing fixative.
iv. Temperature of sample: room temperature/refrigerated • Ideal fixative: 95% ethanol
v. Any evidence of clotting: yes/no. • Stains:
• One Romanowsky stain–Giemsa/May-Grünwald
Equipment and reagents Giemsa or Leishman stain can be performed on the
Essential air‑dried smear and
• Papanicolaou stain performed on the wet‑fixed smears.
• Gloves and masks
• Laboratory centrifuge
Optimal
• Centrifuge tubes: disposable, transparent plastic screw
capped One air‑dried smear and one to two unstained alcohol‑fixed
• Glass marking colored pencil slides may be prepared at the outset  [to attempt
• Applicator sticks/Pasteur pipette with rubber teat immunocytochemistry (ICC) in case] in low‑volume samples
• Clean glass slides where a CB might not be possible.

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IAC effusion fluid cytology guidelines
Processing of hemorrhagic samples
There are several methods of processing hemorrhagic
samples. The laboratory may choose from any technique that
is well‑standardized locally.
Pre‑smearing techniques
• About 10–15 mL of sample is taken in a plastic conical
centrifugation tube. One percent acetic acid (1 mL) is added
for 10 min. The sample is centrifuged at 2000 rpm for 5 min.
The supernatant is decanted. The sediment cells are washed
twice with phosphate‑buffered saline (PBS). Three or more
smears may be prepared from washed cells as above.
Post‑smearing techniques
• In hemorrhagic samples, an extra smear for hemolysis is
prepared.[1‑3]
Hemolysis
Hemolysis can be achieved by any one of the following
methods:
• Carnoy’s fixative
• Glacial acetic acid
• Saline re‑hydration
• Saponin.
Glacial acetic acid
15 mL of sample + 1 mL of 1% glacial acetic acid. Keep at
room temp for 5–10 min. Centrifuge, wash the deposit with
PBS, centrifuge, and make smear with deposit.
Saline re‑hydration technique
This technique is used to lyse red blood cells  (RBCs) in
hemorrhagic fluids. It improves the yield of diagnostic cells,
and thus increases the accuracy and also reduces the eyestrain
while screening.[5]
This technique is simple and cost‑effective, compared with
other techniques, such as Carnoy’s fixative, glacial acetic
acid, and saponin. Normal saline is readily available and needs
no preparation and has an unlimited shelf‑life. However, the
method causes blotting artifacts which may be observed more
in nonepithelial cells.
The underlying principle of this technique is that the physical
damage caused by air‑drying to RBCs is more than that caused
to the epithelial cells (other cells) and the following rehydration
leads to the rupture of the RBCs and simultaneous retention
of the epithelial cells (other cells).
Method
• An extra smear from the remaining sediment is prepared
• The smear is dried in an incubator at 37°C or at room
temperature for 5 min
4 Journal of Cytology  ¦  Volume 37  ¦  Issue 1  ¦  January-March 2020
• The smear should be kept in a horizontal position (preferably
on a staining bar)
• The smear should be rehydrated with normal saline for
30 s
• The saline is carefully and gently poured through one side
of the smear to prevent the washing off
• The saline is blotted by holding the slides vertically
• The smears are fixed immediately.
The important factors for optimal results are as follows:
• Time duration for drying of the smear should not exceed
the optimal time (5 min)
• The smears should not be re‑hydrated for more than
optimal time (30 s) to avoid blurring of cellular features
• The time lag between complete drying and re‑hydration
of smears should be less than 10 min.
Fluid with no/sparse sediment
After centrifugation, if no sediment or very scant sediment
is obtained, the fluid has to be further processed by
cytocentrifugation.
Optional
Liquid‑based cytology preparation
Following centrifugation and preparation of one air‑dried
smear from the sediment, the remaining sediment is fixed
in the manufacturer‑recommended fixative (methanol based
on Thin‑Prep and ethanol based on SurePath). A minimum
of one liquid‑based cytology (LBC) smear is prepared as
per the manufacturer’s protocol, stained with Papanicolaou
stain.[6]
Storage of sample
Essential
After processing, the remaining sample should be
stored in the refrigerator at 2–8°C till the final report is
generated. This stored sample is useful for making repeat
smear if the cellularity is inadequate, for making CB or
ancillary techniques if required. After the reporting, the
stored sample should be discarded as per the biowaste
management policy.
Cell Block
Optimal – Highly desirable
Cell Block (CB) preparation is a technique  (in addition to
smears) of cell concentration without compromising the
cellular content and preserving the tissue architecture.
Preparation of Cell Block from the effusion sediment
is desirable and is being frequently requested by the
clinician
Advantage: The major advantage is in the application
of immunohistochemistry  (IHC) for various situations
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IAC effusion fluid cytology guidelines
including the distinction between reactive mesothelial cells
and adenocarcinoma, mesothelioma versus adenocarcinoma,
characterization and typing of lymphoproliferative disorders
and poorly differentiated malignancies, and to elucidate the
primary site of tumor when occult at presentation.
Other benefits: CBs permit storage of the
cellular material as formalin‑fixed paraffin‑embedded
material that can be used at any later time for molecular
technique such as FISH, sequencing, genomics, and further
research projects.
Methods for preparing Cell Blocks[7‑10]
1. Direct sedimentation
2. Agar embedding
3. Plasma‑thrombin or thromboplastin clot method
Direct sedimentation
This method is best used when a large quantity (>1 mL) of
sediment is obtained.
Equipment and reagents:
1. Fixative: 10% buffered formalin
Formaldehyde solution (40%) 100 mL
Tap water 900 mL
2. Whatman’s No. 1 filter paper/lens paper
3. Tissue cassette
4. Applicator sticks
5. Screw capped disposable plastic transparent centrifuge
tube.
Procedure:
• The sample should be centrifuged.
• The supernatant is discarded.
• An equal volume of fixative is added to the sediment and
it is thoroughly mixed.
• The tube is capped and kept overnight in the refrigerator.
• The fixed sediment is poured onto filter paper/lens paper.
• The sediment is wrapped securely in the filter paper and
placed it into a labeled tissue cassette.
• Subsequently, the cassette is kept in a jar containing
fixative.
• Finally, this is processed similar to a tissue specimen.
Agar embedding
This method is cost‑effective and useful when a reasonably
good quantity of the sediment is obtained (0.5–1 mL).
Equipment and reagents:
1. 4% Agar solution
Dissolve 4 g of bacterial agar in 100 mL of boiling water.
The stock should be stored in 10‑mL aliquots in screw capped
test tubes in the refrigerator. The shelf‑life is best for 2 months.
2. 10% Buffered formalin
3. Whatman’s No. 1 filter paper/lens paper
Journal of Cytology  ¦  Volume 37  ¦  Issue 1  ¦  January-March 2020
4. Tissue cassette
5. Applicator sticks
6. Screw capped disposable plastic transparent centrifuge tube
6. Plastic lid of injection vials to use as mould
7. Pasteur pipette
8. Scalpel
9. Water bath
Procedure:
• The sample is centrifuged and the supernatant is discarded.
• The agar is melted in a water bath.
• With the help of a Pasteur pipette, the molten agar is
dropped into the vial lid.
• A small depression is made in the center of the semisolid
agar.
• With the tip of a Pasteur pipette, the sediment is carefully
taken out little by little and deposited into the depression.
Care should be taken not to lose the sediment into the
lumen of the pipette.
• The sediment deposit is covered and filled with few more
drops of molten agar.
• The agar is allowed to completely solidify.
• With a scalpel, slowly the agar is taken out and mounted (with
intact sediment inside) from the vial lid. Care should be
taken not to break the pellet while removing.
• The pellet is wrapped securely in the filter
paper (pre‑moistened with formalin fixative) and placed
in a labeled tissue cassette.
• The cassette is kept in a jar containing fixative (at least for 4
h) and this material is further processed as a tissue specimen.
A. Plasma‑thrombin clot method
B. Plasma‑thromboplastin clot method[11]
This method is applicable for any quantity of sediment
and is even more useful when the quantity of the sediment
obtained (<0.5 mL) is scant.
A and B. Equipment and reagents
1. 10% buffered formalin
2. Whatman’s No. 1 filter paper/lens paper
3. Tissue cassette
4. Applicator sticks (optional)
5. Screw capped disposable plastic transparent centrifuge
tube
6. Pasteur pipette
7. Thrombin/thromboplastin
8. Pooled plasma.
A. Procedure – suitable when effusion sample is received with
anticoagulant
• The sample is centrifuged and supernatant discarded
• To the cell sediment, 5 mL of 10% buffered formalin
is added and fixed at room temperature for at least 6 h.
Overnight fixation may be done
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IAC effusion fluid cytology guidelines

• The sample is centrifuged and the supernatant is discarded.
• Two washes with PBS are given and the supernatant
is removed as much as possible to leave only the cell
sediment behind
• 100 µL  (two drops) of pooled plasma is added to the
sediment and mixed well
• 50 µL  (one drop) of thrombin/thromboplastin is added
and mixed well again
• The tube is allowed to stand for 5 min to form the clot
• The clot in the test tube is slid onto the pre‑moistened
filter paper and placed in a labeled tissue cassette
• This is processed further as a small biopsy in the tissue
processor.
agar, thrombin, gelatine, and egg albumin. The ideal method
should be simple, faster, reproducible, and able to concentrate
cells in a limited field without loss of cellular material and
cost‑effective.
All traditional methods of CB require overnight formalin
fixation and processing and subsequent manual embedding
similar to histological techniques. This would cause the delay
in diagnosis.
Shandon Cytoblock  (Thermo) and Cellient Automated Cell
Block System  (Hologic) are the two automated cellblock
preparation systems available. For these systems, the
manufacturer’s recommendations should be followed.

B. Procedure  –  suitable when effusion sample is received Reporting of Effusion Cytology

without anticoagulant
Essential components of report
• The sample is centrifuged and the supernatant discarded. Volume:
• Two drops  (100 µL) of pooled plasma is added to the
sediment and mixed well Appearance: mL/L
• One drop (50 µL) of thromboplastin is added and mixed Specimen cellularity: low/moderate/high
well again
• The tube is allowed to stand for 5 min to form the clot. Description (Optional)
• The clot in the test tube is slid onto the filter paper Background: clear/proteinaceous/granular/hemorrhagic
pre‑moistened with formalin fixative
Interpretation: After evaluation of the smears prepared, the
• The sediment is wrapped securely in the filter
case may be placed in any of the five recommended diagnostic
paper (pre‑moistened with formalin fixative) and placed
categories detailed in Table 2.
into a labelled tissue cassette
• The cassette is placed in a jar containing neutral Explanatory note for categories
buffered formalin (fixative) for at least for 4 h or left overnight
Category 1: Unsatisfactory for evaluation
• This is processed further as a small biopsy in the tissue
processor. Smears with no cells for evaluation or those
which show contamination by artefacts, bacterial colony, or those
Technical consideration which show cells that are poorly preserved and show cellular
degenerative changes, and therefore not suitable for interpretation.
1. The pooled plasma can be preserved in a freezer up to
Representative case is illustrated in Figure 1.
1 month in 1‑mL aliquots. Plasma may be extracted from
blood collected in EDTA vial
2. Both thrombin and thromboplastin can be used interchangeably
with “A” and “B” procedures with similar results
3. Store thrombin/thromboplastin in the refrigerator at
2–80°C
4. The reagents should be brought to the room temperature
before processing
5. Care should be taken to use different pipettes to take out
plasma and thrombin/thromboplastin
6. If clot is not formed within 5 min, extra PBS washings are
given and the procedure is repeated using fresh plasma.
7. Check the expiry date of thrombin/thromboplastin vial
before use.

Optional
Automated Cell Block systems[12‑14]
Various methods for preparing CBs have been reported and
the techniques are in a state of continuous improvement.
Different methods include usage of various adjuvants such as
Figure 1: Category 1. Unsatisfactory for evaluation: smear shows cells,
but there is extensive obscuration by crystals of anticoagulant (May–
Grünwald Giemsa stain)

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IAC effusion fluid cytology guidelines
Category 2: No malignant cells detected/benign cellular
changes
This category represents a wide spectrum of cases of effusions in the
absence of cancer. Hence, besides reporting “no malignant cells,”
the report can also describe the cellular changes which can include
the presence of mesothelial cells and inflammatory cells in variable
numbers. The specific diagnoses in this category include (i) reactive
mesothelial proliferation, (ii) acute inflammation,  (iii) chronic
inflammation, (iv) lymphocytic effusion, and (v) specific infections
with organism identified such as cocci, bacilli, mycobacteria,
nocardia, fungus, parasites such as microfilaria, hydatid cyst, or
any other infectious agent. Representative cases are illustrated in
image panels of Figure 2A and B.
Category 3: Atypical cells, NOS
This category represents smears that show cells with
cytological atypia that quantitatively or qualitatively do not
favor malignancy. Representative cases are illustrated in image
panels of Figure 3.
Category 4: Atypical cells, suspicious for malignancy
This category represents smears that show cells with
cytological atypia that quantitatively or qualitatively fall short
of a diagnosis of frank malignancy but are enough to warrant
suspicion of malignancy. Ancillary techniques of ICC are
useful in this setting and are recommended wherever possible.
Representative cases are illustrated in image panels of Figure 4.
Category 5: Malignant cells seen
This category represents smears that show unequivocal
malignant cells. Further categorization into carcinoma and
noncarcinomatous malignancy is warranted. Representative
a b
c d
Figure 2A: Category 2: No malignant cells detected/benign cellular
changes. (a) Mesothelial cells and inflammatory cells predominantly
neutrophils. (b) Predominantly acute inflammatory cells and bile indicating
biliary peritonitis. (c) Microfilaria seen along with mixed inflammatory
cells. (d) Lupus effusion showing neutrophils, macrophages, and lupus
erythematosus (LE) cell (arrow). (a, c, d) May–Grünwald Giemsa
stain; (b) Papanicolaou stain
Journal of Cytology  ¦  Volume 37  ¦  Issue 1  ¦  January-March 2020
cases are illustrated in Figure 5A and B. The different types
of malignancy and the optimal use of ICC are detailed below.
Immunocytochemistry – Optimal[15‑18]
ICC may be performed on CBs or direct smear preparations
or on LBC smears. The list of markers is detailed in Table 3.
For smear preparations, fixatives may be 95% ethanol,
formalin‑alcohol, and acetone‑alcohol.
Adenocarcinoma vs reactive mesothelial cells
Any two mesothelial markers and any two markers for
adenocarcinoma may be chosen from the following list:
(a) Mesothelial markers: calretinin, CK5/6, desmin, D2‑40
(b) Adenocarcinoma markers: BerEP4, MOC31, EMA
Typing of malignant tumors
(a) Mesothelioma: diagnosis of mesothelioma requires
integration of clinicoradiological features with
cytopathological findings, including immunostaining results.
Mesothelial markers: calretinin, CK5/6, WT1, D2‑40 (positivity
for any two markers is desirable, Table 3)
Strong diffuse membranous EMA positivity is also a feature
of mesothelioma
(a) Nonmesothelial neoplasms: carcinomas and other
malignant neoplasms – see Table 3.[19‑21]
a b
c d
e f
Figure 2B: Category 2: No malignant cells detected/benign cellular
changes. Paired images of three cases showing variable admixture of
reactive mesothelial cells and inflammatory cells, mostly lymphocytes. (a,
c, e) May–Grünwald Giemsa stain; (b, d, f) Papanicolaou stain
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IAC effusion fluid cytology guidelines

Table 2: IAC Diagnostic Categories for Reporting Serous Effusion Cytology samples
IAC reporting category Cytopathology Diagnosis Remarks
1 Unsatisfactory for evaluation No cells seen/Obscuration by blood, artifacts, extensive degenerative changes
2A No malignant cells detected Correlate clinically and with imaging studies and microbiological studies
B Benign Changes seen
Reactive mesothelial cells
Inflammatory cells seen
Lymphocyte‑rich effusion
Specific infections
Tuberculosis, Microfilaria, Fungal
infection, Hydatid cyst, any other
3 Atypical cells, not otherwise Repeat Cytology
specified (NOS) Correlate clinically and with imaging studies
Ancillary techniques‑Optional
4 Atypical cells, suspicious for Repeat Cytology evaluation
malignancy Ancillary techniques‑Optimal/essential
5 Malignant cells seen (of mesothelial Subtype the malignancy wherever possible on cytomorphology and ancillary
or non‑mesothelial origin) techniques of immunocytochemistry

a b a b
Figure 3: Category 3: Atypical cells, NOS. Paired images showing loose
aggregate of cells showing nuclear atypia that fall short of a diagnosis
of malignancy. (a) May–Grünwald Giemsa stain; (b) Papanicolaou stain

(b) Reactive Mesothelial proliferation and distinction from

adenocarcinoma
BerEP4, MOC31, and EMA positivity favors adenocarcinoma;
calretinin/desmin positivity favors reactive mesothelial proliferation
(c) Reactive mesothelium versus mesothelioma
Strong diffuse membranous EMA and loss of BAP1 and MTAP
on IHC with or without p16 deletion on FISH are diagnostic of
mesothelioma. Desmin positivity favors reactive mesothelium.[22]
Note: While it is important to use suggested panel given above,
individual laboratory must validate suitable panel according to
their setup and decide which panel works optimum in their setting.
Lung cancer specimen – Adenocarcinoma lung:
Optional[23,24]
Malignant pleural effusion sample is a commonly received
sample in advanced stage lung adenocarcinoma, and after
confirming the lung primary, the cytopathologists are expected
to do other ancillary tests, which are required for treatment
decision‑making.
Samples
Cell sediment from the effusion, residual sediment after LBC
preparation, and direct scrape from an air‑dried/H  and  E/
c d
Figure 4: Category 4: Atypical cells, suspicious for malignancy. Paired
images of two cases showing mesothelial cells admixed with loose
aggregate of cells (a, b) or dispersed cells (c, d) showing nuclear atypia
that quantitatively or qualitatively fall short of a diagnosis of malignancy
but are enough to warrant suspicion of malignancy. (a, c) May–Grünwald
Giemsa stain; (b, d) Papanicolaou stain
Papanicolaou‑stained cellular representative smear/CBs are
all suitable specimens for molecular testing.
For IHC, CBs are preferred. If unavailable, smears may be used
but only in laboratories that have the method standardized/
validated on smears.
Tests done: EGFR mutation; ALK and ROS 1 rearrangements
and PDL1 expression.
For molecular testing
Samples that are of adequate cellularity and with at least 20%
tumor content may be shipped to the molecular laboratory for
further testing.
DNA extraction may be carried out from any of the samples
from a commercially available DNA extraction kit.

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IAC effusion fluid cytology guidelines

Table 3: Immunocytochemical markers useful in Effusion Cytology

Diagnosis Calretinin D2‑40 WT‑1 TTF‑1 CK7 CK20 PAX8 SYN/ MOC‑31 GATA3 P63/
CHR EMA P40
Mesothelioma + + + ‑ + ‑ ‑ ‑ EMA+ + ‑
MOC31‑
Small cell carcinoma ‑ ‑ ‑ + ‑ ‑ ‑ + ‑ ‑ ‑
Squamous cell carcinoma ‑ ‑ ‑ ‑ +/‑ ‑ ‑ ‑ ‑ + +
Adenocarcinoma, lung origin ‑ ‑ ‑ + + ‑ ‑ ‑ + ‑ ‑
Adenocarcinoma, ovary origin ‑ ‑ + ‑ + ‑ + ‑ + ‑ ‑
Adenocarcinoma, breast origin ‑ ‑ ‑ ‑ + ‑ ‑ + + ‑
Adenocarcinoma, stomach origin ‑ ‑ ‑ ‑ + ‑ ‑ ‑ + ‑ ‑
Adenocarcinoma, colorectal origin ‑ ‑ ‑ ‑ ‑ + ‑ ‑ + ‑ ‑
Germ cell tumours SALL4+ Negative for all mesothelial and epithelial markers
Lymphoma CD45+ Negative for all mesothelial and epithelial markers
Note: ICC marker panel must be chosen based on the cytomorphological diagnosis or differential diagnosis in the clinical context. Any 2 expected positive
and expected negative markers may be chosen based on the differential diagnosis. p40 is more specific than p63 for squamous cell carcinoma. PAX8
positivity is seen not only in mullerian origin tumours, but also in renal, thyroid and thymic tumors. CDX2 is useful for confirming colorectal origin. p53 is
useful for confirming mesothelioma and high-grade serous carcinoma of ovarian origin. GATA-3 is positive in subset of mesothelioma and squamous cell
carcinomas in addition to majority of breast and urothélial carcinomas.[19] Small cell carcinomas (pulmonary and extrapulmonary) are positive for TTF-1 in
40 to over 90% cases[20,21]

a b a b

c d c d

e f
Figure 5A: Category 5: Malignant cells seen, non-mesothelial origin. (a‑d)
Paired images of two cases showing malignant cells of adenocarcinoma
type; (a, b) numerous papillaroid aggregates of malignant cells; (c, d)
dispersed malignant cells in a background of mucin. (e, f) Paired image
of small cell carcinoma. (a, c, e) May–Grünwald Giemsa stain; (b, d, f)
Papanicolaou stain
EGFR mutation testing
e f
Figure 5B: Categor y 5: Malignant cells seen, mesothelial
origin – mesothelioma. (a,b) Three‑dimensional papillary aggregates
of malignant cells; (c) cell block; (d‑f) immunocytochemistry on
the cell block. MOC31 and TTF1 are negative, whereas calretinin
is strongly positive and WT1 shows patchy positivity in the
malignant cells confirming mesothelioma. (a) May–Grünwald Giemsa
stain, (b) Papanicolaou stain, (c) hematoxylin–eosin stain, (d‑f)
immunoperoxidase stain

Testing for EGFR driver mutations in exons 18, 19,

20, and 21 can be done by real‑time polymerase chain Testing for ALK rearrangement
reaction (PCR)‑based methods using commercially available ICC on CBs can be done for ALK testing using D5F3 Ventana
kits, next‑generation sequencing, or digital PCR. automated IHC assay.

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IAC effusion fluid cytology guidelines

Table 4: Sample reporting format (1) Conclusion

Name: ABC………… Age/Sex: 50/Female The IAC recommendations and guidelines for receipt,
Hospital registration number: 123456 preparation, and reporting of serous effusion cytology samples
Procedure: Left paracentesis are expected to be followed by all accredited laboratories to
Date and time of collection: Nov 30, 2019. 10AM ensure quality in laboratory cytopathology practice which in
Anticoagulant used or not, including type: Heparin turn will have an impact on patient care and management in
Specimen type: Ascitic fluid Quantity received: 40mL clinical practice.
Gross appearance: Hemorrhagic
No. of smears evaluated: Two Guidelines Committee invited members
Microscopy: Amit Adhya, Asitava Mondal, Bidyut Goswami, Gulab Gupta,
Cellularity: Low/Moderate/High√ (Tick one) Madhu Mati Goel, Malathi M, Manju Kaushal, Manjula Jain,
Adequacy: Satisfactory for interpretation Meherbano Kamal, Nuzhat Husain, Pavneet Selhi, Ranjan
Cells: Malignant cells, mesothelial cells and inflammatory cells seen.
Agrawal, Ravi Mehrotra, Reena Bharadwaj, RGW Pinto,
Background: hemorrhagic
Rohit Tewari, Ronica Baruah, Shailaja Shukla, Shubhada
Cellular Description (optional):
Cell Block made: Yes
Kane, Shyama Jain, Siddaraju Raju, Surendra Kumar Verma,
Diagnostic Category: Category 5, Malignant cells seen. Uma Handa.
Subtype: Adenocarcinoma
Financial support and sponsorship
Comment: Immunocytochemistry for further typing of
adenocarcinoma if required may be performed.
Indian Academy of Cytologists.
Conflicts of interest
There are no conflicts of interest.
Table 5: Sample reporting format (2)
Name: KLM………… Age/Sex: 30/Male
Hospital registration number: 567890
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IAC effusion fluid cytology guidelines

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