Indian Academy of Cytologists Guidelines for Collection, Preparation, Interpretation, and Reporting of Serous Effusion Fluid Samples.

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. Copyright:

INTRODUCTION

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. To achieve uniformity in this process across our country, the 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:

ESSENTIAL: these are absolute and non-negotiable recommendations, and if a laboratory cannot achieve this, then the sample may be referred to a center which is equipped to fulfil these criteria.

OPTIMAL: this is achievable in any good laboratory and should be part of the protocol and can be made mandatory for accreditation.

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

Essential

Clinical details

The sample should accompany a requisition form with the following necessary details [Table 1]:

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

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

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 fluid, pericardial fluid, peritoneal washing, and rarely pleural 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.[34]

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 anticoagulant prevents clotting of sample and can provide accurate cell counts if required and also achieves optimal smear quality.

The choice of anticoagulants are as follows:

Heparin easily available in OPDs/wards – 3 units/mL (can be titrated depending on the strength of heparin). Usually, the syringe used for paracentesis or collection container is rinsed in heparin.

Citrate

EDTA – ethylenediaminetetraacetic acid

Ammonium oxalate – 1% to be added in a ratio of nine parts fluid to one part anticoagulant. This is a cheap option

Note: NO fixative should be added to the fluid. Formalin prevents cells from adhering to the slide and interferes with the quality of Papanicoloau staining. Alcohol should not be added as it causes precipitation of proteins and thus interferes with the adherence of the cells to slide.

CYTOPREPARATION OF EFFUSION SAMPLES

Receipt of sample in cytopathology laboratory

Essential

Upon receipt of the sample, the laboratory generates its accession number as per individual laboratory/hospital policy and the sample is accepted for processing after checking of patient identifiers on the request form and on the sample label.

Optimal

Barcoding of sample containers is a common practice.

Gross evaluation

Essential

Gross or macroscopic evaluation must be performed by the laboratory technician/cytotechnician and the following points are to be noted:Quantity – mL/L

Color – clear/straw-colored/yellow/brown/red/chylous/purulent/hemorrhagic/other (please specify)

Consistency: serous/mucoid/gelatinous/thick copious/tar-like/other

Temperature of sample: room temperature/refrigerated

Any evidence of clotting: yes/no.

Equipment and reagents

Essential

Gloves and masks

Laboratory centrifuge

Centrifuge tubes: disposable, transparent plastic screw capped

Glass marking colored pencil

Applicator sticks/Pasteur pipette with rubber teat

Clean glass slides

Cover slips

A wide-mouth specimen bottle with fixative

Normal saline

Glass marking diamond pencil

Mayer's egg albumin.

Optimal

Biosafety cabinets

Cytocentrifuge machine, funnels, clips, and blotting cards

Applicator sticks with thin tightly wound cotton

Positively charged slides.

Processing

Essential

Technician handling the fluid specimen should wear adequate personal protective gear.

Centrifuge all samples with a desktop laboratory centrifuge.

In case a fibrin clot is formed, it should be thoroughly smashed with an applicator stick. In case a large clot remains, it may be processed as CB.

A representative volume of the fluid (10–15 mL) should be centrifuged using capped plastic tubes, 2000 rpm for 10 min.

Subsequently, most of the supernatant should be gently decanted/removed. Care should be taken not to disrupt the intact sediment while decanting, as it may require repeat centrifugation. From the sediment, smears are made.

A small volume of the sediment using an applicator/disposable Pasteur pipette should be placed onto a pre-labeled albuminized slide. Thereafter, the sediment should be spread gently using the flat surface of another glass slide or optimally by rolling the cotton tipped applicator stick gently in a rotatory motion on the slide, so as to make a thin evenly spread smear. Note that the cotton tipped applicator should be moistened by the last drop of fluid at the time of decantation before picking up the sediment for good preparation.

A minimum of two smears should be prepared from each sample – one is air-dried and another is wet-fixed/fixed immediately in a coplin jar/jar containing fixative.

Ideal fixative: 95% ethanol

Stains:

One Romanowsky stain–Giemsa/May-Grünwald Giemsa or Leishman stain can be performed on the air-dried smear and

Papanicolaou stain performed on the wet-fixed smears.

Optimal

One air-dried smear and one to two unstained alcohol-fixed slides may be prepared at the outset [to attempt immunocytochemistry (ICC) in case] in low-volume samples where a CB might not be possible.

Processing of hemorrhagic samples

There are several methods of processing hemorrhagic samples. The laboratory may choose from any technique that is well-standardized locally.

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.

In hemorrhagic samples, an extra smear for hemolysis is prepared.[123]

Hemolysis

Hemolysis can be achieved by any one of the following methods:

Glacial acetic acid

Sline re-hydration

Saponin.

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.

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

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 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[78910]

Direct sedimentation

Agar embedding

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:

Fixative: 10% buffered formalin

Formaldehyde solution (40%) 100 mL

Tap water 900 mL

Whatman's No. 1 filter paper/lens paper

Tissue cassette

Applicator sticks

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:

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.

10% Buffered formalin

Whatman's No. 1 filter paper/lens paper

Tissue cassette

Applicator sticks

Screw capped disposable plastic transparent centrifuge tube

Plastic lid of injection vials to use as mould

Pasteur pipette

Scalpel

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[

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

10% buffered formalin

Whatman's No. 1 filter paper/lens paper

Tissue cassette

Applicator sticks (optional)

Screw capped disposable plastic transparent centrifuge tube

Pasteur pipette

Thrombin/thromboplastin

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

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.

B. Procedure – suitable when effusion sample is received without anticoagulant

The sample is centrifuged and the supernatant discarded.

Two drops (100 μL) of pooled plasma is added to the sediment and mixed well

One drop (50 μL) of 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 filter paper pre-moistened with formalin fixative

The sediment is wrapped securely in the filter paper (pre-moistened with formalin fixative) and placed into a labelled tissue cassette

The cassette is placed in a jar containing neutral buffered formalin (fixative) for at least for 4 h or left overnight

This is processed further as a small biopsy in the tissue processor.

Technical consideration

The pooled plasma can be preserved in a freezer up to 1 month in 1-mL aliquots. Plasma may be extracted from blood collected in EDTA vial

Both thrombin and thromboplastin can be used interchangeably with “A” and “B” procedures with similar results

Store thrombin/thromboplastin in the refrigerator at 2–80°C

The reagents should be brought to the room temperature before processing

Care should be taken to use different pipettes to take out plasma and thrombin/thromboplastin

If clot is not formed within 5 min, extra PBS washings are given and the procedure is repeated using fresh plasma.

Check the expiry date of thrombin/thromboplastin vial before use.

Optional

Automated Cell Block systems[121314]

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 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.

REPORTING OF EFFUSION CYTOLOGY

Essential components of report

Volume:

Appearance: mL/L

Specimen cellularity: low/moderate/high

Description (Optional)

Background: clear/proteinaceous/granular/hemorrhagic

Interpretation: After evaluation of the smears prepared, the case may be placed in any of the five recommended diagnostic categories detailed in Table 2.

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
2 A	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 specified (NOS)	Repeat Cytology
		Correlate clinically and with imaging studies
		Ancillary techniques-Optional
4	Atypical cells, suspicious for malignancy	Repeat Cytology evaluation
		Ancillary techniques-Optimal/essential
5	Malignant cells seen (of mesothelial or non-mesothelial origin)	Subtype the malignancy wherever possible on cytomorphology and ancillary techniques of immunocytochemistry

Explanatory note for categories

Category 1: Unsatisfactory for evaluation

Smears with no cells for evaluation or those which show contamination by artefacts, bacterial colony, or those which show cells that are poorly preserved and show cellular degenerative changes, and therefore not suitable for interpretation. Representative case is illustrated in Figure 1.

Figure 1

Category 1. Unsatisfactory for evaluation: smear shows cells, but there is extensive obscuration by crystals of anticoagulant (May–Grünwald Giemsa stain)

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.

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

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

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.

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

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.

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

Category 5: Malignant cells seen

This category represents smears that show unequivocal malignant cells. Further categorization into carcinoma and noncarcinomatous malignancy is warranted. Representative cases are illustrated in Figure 5A and B. The different types of malignancy and the optimal use of ICC are detailed below.

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

Figure 5B

Category 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

Immunocytochemistry – Optimal[15161718]

ICC may be performed on CBs or direct smear preparations or on LBC smears. The list of markers is detailed in Table 3.

Table 3

Immunocytochemical markers useful in Effusion Cytology

Diagnosis	Calretinin	D2-40	WT-1	TTF-1	CK7	CK20	PAX8	SYN/ CHR	MOC-31 EMA	GATA3	P63/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[2021]

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:

Mesothelial markers: calretinin, CK5/6, desmin, D2-40

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

Nonmesothelial neoplasms: carcinomas and other malignant neoplasms – see Table 3.[192021]

Reactive Mesothelial proliferation and distinction from adenocarcinoma

BerEP4, MOC31, and EMA positivity favors adenocarcinoma; calretinin/desmin positivity favors reactive mesothelial proliferation

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[2324]

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/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.

EGFR mutation testing

Testing for EGFR driver mutations in exons 18, 19, 20, and 21 can be done by real-time polymerase chain reaction (PCR)-based methods using commercially available kits, next-generation sequencing, or digital PCR.

Testing for ALK rearrangement

ICC on CBs can be done for ALK testing using D5F3 Ventana automated IHC assay.

Testing for ROS 1 rearrangement

ICC on CBs can be done for ROS 1 testing using clone D4D6 (Cell Signaling Technology, Danvers, MA, USA). All positive reactions are confirmed by ROS1 FISH.

Testing for PD-L1 expression

ICC on CB can be done using SP263 clone (Ventana), 28-8 (Dako), or 22C3 (Dako) clone on Ventana or DAKO platforms, respectively, unless validated otherwise.

Breast cancer in Effusion

Further molecular testing for ER, PR and HER2/neu on cell blocks of effusion sample may be performed.

Reporting templates and sample reports are shown in Tables 4 and 5.

Table 4

Sample reporting format (1)

Name: ABC…………     Age/Sex: 50/Female

Hospital registration number: 123456

Procedure: Left paracentesis

Date and time of collection: Nov 30, 2019. 10AM

Anticoagulant used or not, including type: Heparin

Specimen type: Ascitic fluid     Quantity received: 40mL

Gross appearance: Hemorrhagic

No. of smears evaluated: Two

Microscopy:

Cellularity: Low/Moderate/High√ (Tick one)

Adequacy: Satisfactory for interpretation

Cells: Malignant cells, mesothelial cells and inflammatory cells seen.

Background: hemorrhagic

Cellular Description (optional):

Cell Block made: Yes

Diagnostic Category: Category 5, Malignant cells seen.

Subtype: Adenocarcinoma

Comment: Immunocytochemistry for further typing of adenocarcinoma if required may be performed.

Table 5

Sample reporting format (2)

Name: KLM…………      Age/Sex: 30/Male

Hospital registration number: 567890

Procedure: Left thoraacocentesis

Date and time of collection: Nov 30, 2019. 10AM

Anticoagulant used or not, including type: Ammonium oxalate

Specimen type: Pleural fluid      Quantity received: 20 mL

Gross appearance: Pale yellow

No. of smears evaluated: Two

Microscopy:

Cellularity: Low/Moderate√/High

Adequacy: Satisfactory for interpretation

Cells: Mesothelial cells and inflammatory cells seen. The inflammatory cells are mostly lymphocytes

Background: Fluidy

Cellular Description (optional):

Cell Block made: No

Diagnostic Category: Category 2, No Malignant cells seen.

Benign- Lymphocyte-rich effusion

CONCLUSION

The IAC recommendations and guidelines for receipt, preparation, and reporting of serous effusion cytology samples are expected to be followed by all accredited laboratories to ensure quality in laboratory cytopathology practice which in turn will have an impact on patient care and management in clinical practice.

Guidelines Committee invited members

Amit Adhya, Asitava Mondal, Bidyut Goswami, Gulab Gupta, Madhu Mati Goel, Malathi M, Manju Kaushal, Manjula Jain, Meherbano Kamal, Nuzhat Husain, Pavneet Selhi, Ranjan Agrawal, Ravi Mehrotra, Reena Bharadwaj, RGW Pinto, Rohit Tewari, Ronica Baruah, Shailaja Shukla, Shubhada Kane, Shyama Jain, Siddaraju Raju, Surendra Kumar Verma, Uma Handa.

Financial support and sponsorship

Indian Academy of Cytologists.

Conflicts of interest

There are no conflicts of interest.