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| Year : 2020 | Volume
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| Issue : 1 | Page : 1-11 |
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| Indian academy of cytologists guidelines for collection, preparation, interpretation, and reporting of serous effusion fluid samples |
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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
Click here for correspondence address and email
| Date of Submission | 02-Dec-2019 |
| Date of Decision | 04-Dec-2019 |
| Date of Acceptance | 05-Dec-2019 |
| Date of Web Publication | 23-Dec-2019 |
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 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
How to cite this article:
Srinivasan R, Rekhi B, Rajwanshi A, Pathuthara S, Mathur S, Jain D, Gupta N, Gautam U, Rai N, Nijhawan VS, Iyer V, Dey P, Deb P, Prasoon D. Indian academy of cytologists guidelines for collection, preparation, interpretation, and reporting of serous effusion fluid samples. J Cytol 2020;37:1-11 |
How to cite this URL:
Srinivasan R, Rekhi B, Rajwanshi A, Pathuthara S, Mathur S, Jain D, Gupta N, Gautam U, Rai N, Nijhawan VS, Iyer V, Dey P, Deb P, Prasoon D. Indian academy of cytologists guidelines for collection, preparation, interpretation, and reporting of serous effusion fluid samples. J Cytol [serial online] 2020 [cited 2023 Mar 28];37:1-11. Available from: https://www.jcytol.org/text.asp?2020/37/1/1/273793 |
Effusion Guidelines Committee of IAC
Guidelines drafting and finalization committee
| 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]:
- 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.[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 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.
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],[2],[3]
Hemolysis
Hemolysis can be achieved by any one of the following methods:
- Carnoy's fixative
- Glacial acetic acid
- Sline 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
- 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[7],[8],[9],[10]
- 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
[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
- 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[12],[13],[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 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
Click here to view |
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)
Click here to view |
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 2]A and [Figure 2] 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]. | Figure 3: Category 3: Atypical cells, NOS. Paired images showing loose aggregate of cells showing nuclear atypia that fall short of a diagnosisof malignancy. (a) May–Grünwald Giemsa stain; (b) Papanicolaou stain
Click here to view |
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 looseaggregate of cells (a, b) or dispersed cells (c, d) showing nuclear atypiathat quantitatively or qualitatively fall short of a diagnosis of malignancybut are enough to warrant suspicion of malignancy. (a, c) May–GrünwaldGiemsa stain; (b, d) Papanicolaou stain
Click here to view |
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 5]A and [Figure 5]B. The different types of malignancy and the optimal use of ICC are detailed below.
Immunocytochemistry – Optimal[15],[16],[17],[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:
- 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].[19],[20],[21]
- 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[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/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 [Table 4] and [Table 5].
| 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.
Indian Academy of Cytologists.
Financial support and sponsorship
Indian Academy of Cytologists.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Dr. Radhika Srinivasan
Secretary, Indian Academy of Cytologists
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/JOC.JOC_157_19
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5] |
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