Processing and Reporting of Cytology Specimens from Mediastinal Lymph Nodes Collected using Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration: A State-of-the-Art Review

Abstract

Endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) is presently the preferred modality for sampling mediastinal lymph nodes. There is an unmet need for standardization of processing and reporting of cytology specimens obtained by EBUS-TBNA. The manuscript is a state-of-the-art review on the technical aspects of processing and reporting of EBUS-TBNA specimens. A literature search was conducted using the PubMed database, and the available evidence was discussed among the authors. The evidence suggests that at least one air-dried and one alcohol-fixed slide should be prepared from each lymph node pass. The remaining material should be utilized for microbiological analysis (in saline) and cell block preparation (10% formalin or other solutions). Wherever available, rapid-onsite evaluation should be performed to assess the adequacy of the sample and guide the need for additional material. The lymph node aspirate should also be collected in Roswell Park Memorial Institute solution in cases where lymphoma is under consideration. The use of liquid-based cytology provides good quality specimens that are free from blood and air-drying artifacts and can be used wherever available. Sample adequacy and the diagnostic category should be furnished separately in the cytology report.

INTRODUCTION

Endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) is currently the preferred modality for the evaluation of mediastinal lymphadenopathy.[1] It is a minimally invasive technique that provides real-time ultrasonographic guidance for sampling the mediastinal lymph nodes. The diagnostic yield of EBUS-TBNA ranges from 50%–90% at different centers.[2,3,4,5,6,7,8,9,10,11] The variable diagnostic yield is due to different etiologies of enlarged lymph nodes, the type of needle used, the number and size of the lymph nodes sampled, the number of passes obtained from each lymph node station, the availability of rapid-onsite cytological evaluation (ROSE), and the expertise of the operator.[3,4,5,6,7,12,13] The diagnostic yield could also vary depending on the method used to collect, process, and report the cytological specimens.[14,15,16] There is an unmet need for standardization of sample processing technique and reporting of the cytology samples obtained by EBUS-TBNA. While several reviews and statements are available on sample processing of the EBUS aspirate, few have been framed jointly by both clinicians and cytopathologists.[17,18] In this review, we describe the technical knowhow, processing, and reporting of cytology specimens collected by EBUS-TBNA.

The relevant questions concerning different aspects of processing and reporting of cytology specimens obtained using EBUS-TBNA were framed. A systematic review of the PubMed database was performed. We then gathered the evidence pertinent to each question [Table 1],[19,20] and practice points were assigned to each question.

Table 1.

Categorization of the level of evidence

Classification of level of evidence
Level I	High-quality evidence supported by findings from well-executed randomized controlled trials, or unequivocal evidence from well conducted observational studies with strong effects
Level II	Moderate-quality evidence from randomized trials or from several observational studies with some limitations (inconsistency, indirectness, flaws in conduct, reporting bias, imprecise estimates, small sample size, or others)
Level III	Low-quality evidence from observational studies or from controlled trials with serious limitations
Useful Practice Point (UPP)	Not supported by sufficient evidence; however, a consensus reached by the working group, based on clinical experience and expertise

Adapted from Agarwal et al.[20]

1. PROCESSING OF CYTOLOGY SPECIMENS

1.1 What is the ideal method of preparing slides with the aspirate obtained during EBUS-TBNA? How are air-dried and alcohol-fixed slides prepared?

Proper collection and processing of cytologic specimens are crucial for an accurate cytopathological diagnosis.[14,18] The lymph node aspirate obtained by EBUS-TBNA is submitted to direct smear preparation on glass slides. The slides are then either air-dried or immersed in ethyl alcohol.[21,22] Slides dipped in 95% ethyl alcohol are referred to as wet-fixed.[23] The slides should be immediately immersed in the “fixing” solution to avoid any air-drying that will hamper the use of stain. The wet slides get permanently fixed and preserve the shape and structure of the cell. The type of fixative depends upon the stain that will be used subsequently. Many practitioners routinely use both Papanicolaou and Romanowsky stains.[24] The two staining methods are complementary to each other. As Papanicolaou stain is the most widely employed technique, ethyl alcohol is the commonest fixative used. The main advantages of Papanicolaou stain include excellent nuclear chromatin detail and the orangeophilic staining of cytoplasmic keratin. The immersion of slide in liquid fixative, however, results in cell loss. To overcome this cell loss, slides are also air-dried. The cells adhere better to the glass slides when air-dried. Air-dried slides are suitable for Romanowsky stain and the rapid stains (for an onsite evaluation of the specimen). In addition, air-drying of the specimen results in larger cells and preservation of cytoplasm and extracellular matrix compared to the paired alcohol-fixed smear.[24] The air-dried slides can also be rehydrated and stained with the Papanicolaou method. Other methods that can help in better adherence of the cells to the slide include the use of frosted glass slides, charged glass slides, and slides precoated with egg albumin.[23]

There are several methods of smear preparation, including the squash (or the mash) method, the pull-apart method, the one-drop method, the loop method, the press method, and others.[23,25,26] However, there is no head-to-head trial comparing the effect of the method of smear preparation on the diagnostic yield. The primary objective of smear preparation is to obtain a monolayer of cells, and any method can be used according to the ease and preference of a center.[25] The press method is the preferred technique at the authors' center.

Press method: Direct smears are made by applying a drop of the aspirate onto a glass slide (near the specimen label end). Care should be taken to avoid the needle tip of EBUS-TBNA touching the slide. Another person should hold the tip of the TBNA needle with the beveled end facing downwards and guide the expelled material onto the slide. It is advisable to use a shield slide held at an angle of 45°–90° to prevent expelled material from getting splayed. The smear is made by placing a second clean slide directly over the specimen, which is then gently pressed to allow even spread of the material.[14,25] As the aspirate starts to spread, the slide is then gently moved along the length of the slide (feather technique). The smear prepared will be oval/tongue-shaped. This smear can be immediately fixed in an alcohol solution or allowed to dry at room temperature. Alternatively, the aspirated material can be expelled directly into the preservative solution for subsequent slide preparation in the laboratory (liquid-based cytology).[14]

Practice point: The method of preparing slides should be standardized at each center with an aim to obtain a monolayer of cells.[evidence level, UPP]

1.2 How many slides (air-dried and alcohol-fixed) should be made per pass?

Although there is no evidence for the number of slides that should be prepared from each lymph node pass, we believe that at least one air-dried and one wet-fixed slide should be prepared from each lymph node pass. The practice of making a large number of slides per pass (up to 41 slides per pass in one study) was not found to be better than preparing only a pair of slides from each needle pass.[27] Further, the large number of slides would only “exhaust” the laboratory and human resources.

For lung cancer staging, the sampling is performed from the highest lymph node station (N3), followed by N2 and N1 lymph node station. The slides prepared from each lymph node station should be labeled appropriately and sent separately. Some centers use separate needles for each lymph node station during the staging of lung cancer.[28,29,30] However, the needle is expensive, and thus many centers, including ours, reuse the needle.[31,32,33] The sampling is started from the highest lymph node station (N3) followed by N2 and N1.

Practice points:

• At least one air-dried and one wet-fixed slide should be made from each lymph node pass.[evidence level, UPP]

• The practice of preparing many slides should be avoided.[evidence level, UPP]

• For lung cancer staging, slides prepared from each lymph node station should be labeled and sent separately.[evidence level, UPP]

• For lung cancer staging, the same needle can be used, if sampling is performed from the highest to the lowest lymph node station.[evidence level, II]

1.3 What is a cell block, and how should it be made?

A cell block is a technique to convert cytological aspirate into a mini-biopsy, which is then processed, sectioned, stained, and viewed under the microscope just like a histological specimen.[34,35] It can be used to assess the architectural details, and additional sections can be used for special stains, immunohistochemistry, and molecular diagnostic studies.[18] The preparation of a cell block involves three steps, namely, rinsing of the aspirated material, fixing the sample, and making a pellet or a clot.[36]

There are numerous liquid media that can be used for collecting needle aspirate. These include normal saline, 95% alcohol, cell culture solutions (Roswell Park Memorial Institute [RPMI] or Hank balanced salt solution), or fixatives such as Cytolyt (alcohol-based, Cytyc Corporation, Marlborough, MA) or 10% formalin. The choice of solution depends on the clinical diagnosis and the type of investigation needed. Normal saline provides flexibility for using the aspirate for microbiology, cell cultures, flow cytometry, and LBC. However, cellular integrity is compromised within a few hours. The RPMI solution is ideal for supporting cultures and flow cytometry and, hence, is preferred when the clinical suspicion is that of lymphoma. However, it is not readily available at all centers (especially in resource-constrained settings) and must be refrigerated. The most widely used fixative is 10% buffered formalin. It is readily available, inexpensive, and the use of 10% formalin has been validated for the performance of special stains, immunohistochemistry, and molecular analysis. In cases of lung cancer, fixatives with hard metals or harsh acids (e.g. Zenker, B5, B plus, acid zinc formalin, Bouin's solution) should be avoided.[34,37] Heavy metals like mercury interfere with DNA polymerases used in PCR techniques, and harsh acids cause nucleic acid fragmentation.[34,37]

For clot preparation, different agents used are plasma and thrombin, agar, and HistoGel (Thermofisher Scientific Richard-Allan Scientific, Waltham, MA). When samples are transferred to saline or RPMI, plasmin or thrombin is used as the clotting agent. For samples directly transferred to a solution containing formalin or Cytolyt, the clotting agent used is either agar or HistoGel. Another technique used to make the cell block is the collodion bag technique. In this technique, the aspirate is placed in a polymer-linked conical test tube. The polymer bag traps the cellular material, which can be pulled out of the test tube.[36] In a recent study comparing three commonly used cellblock preparation techniques, the collodion bag technique was found to be the best for cellularity, architectural details, and cell preservation compared to needle aspirate transferred in saline (clotted with plasma and thrombin) or formalin (clotted with HistoGel).[38] However, there was no difference between the saline or the formalin technique.[39] Another study compared the normal saline rinse cell block with the tissue coagulum clot method of making the cell block. The EBUS aspirate was expelled over a filter paper using the stylet with the tip of the needle moved circularly to make a cone of tissue.[40] The material was allowed to air dry and then gently pushed in the formalin with filter paper wrapped around it. With the tissue coagulum technique, the diagnostic yield was higher both for lymph node samples and the lung specimens compared to the conventional saline rinse technique.[40] More studies are needed to confirm these findings. The other methods used for making cell block are the rapid cell block method and the automated cell block method.[37]

Flow cytometry is an ancillary technique that is performed on EBUS-TBNA aspirates to immunophenotype lymphomas.[41] The outcome of the flow cytometry depends on the storage material used and the time to processing. The commonly used storage material includes RPMI solution, Hanks' balanced salt solution (HBSS), and Dulbecco's modified eagle's medium (DMEM). In a study evaluating the best storage material for flow cytometry of lymph node aspirates, RPMI solution was found to be the best.[42] The lymph node aspirate had the least nonviable lymphocytes when the aspirate was stored in the RPMI solution at 4°C for 24 h. Moreover, the aspirate could be used for flow cytometry for up to 5 days when stored at 4°C.[42] Other commercially available storage materials include Transfix and phosphate-buffered solution. Material sent in RPMI solution can also be used to perform karyotyping and fluorescent in situ hybridization (FISH).[15] For molecular assessment and mutation analysis, material from both cell block and LBC can be used.[37,43,44]

Practice-points:

• Wherever available, the collodion bag technique may be preferred over the normal saline or the formalin technique for making a cell block.[evidence level, II]

• Tissue coagulum technique is simple and has a higher diagnostic yield and may be used for making cell block.[evidence level, II]

• Normal saline or 10% buffered formalin is easily available and may be used for making cell block.[evidence level, II]

• For lung cancer staging, solutions containing harsh acids or heavy metals should not be used.[evidence level, UPP]

• In case of clinical suspicion of lymphoma, the aspirated material should be placed in RPMI (preferred), HBSS, DMEM, or Transfix solution.[evidence level, II]

• For flow cytometry, the material should be stored at 4°C, and the flow cytometric analysis should be ideally performed within 24 h of collecting the EBUS-TBNA sample.[evidence level, II]

1.4 Should cell block be routinely prepared?

In several studies comparing cytological smear versus cell block method in the evaluation of other body fluids, the addition of cell block increased the diagnostic yield by 10%–15%.[45,46,47] However, there is limited data on the additional utility of cell block for EBUS-TBNA samples.[48,49,50] In a study, cell block preparation from EBUS-TBNA samples increased the diagnostic yield by 7% and provided material for genetic analysis in 60% of the patients with metastatic disease.[48] In another study, the addition of cell block to smear examination increased the diagnostic yield of EBUS-TBNA from 88.8% to 99.6%.[50]

Practice-point: Cell blocks should be made routinely in addition to slides.[evidence level, III]

1.5 How should samples be sent for microbiological investigations?

The EBUS-TBNA aspirate is routinely sent for microbiological investigations. For microbiological cultures, the aspirated material is expelled directly into a sterile container containing normal saline.[18,51] The common investigations ordered are mycobacterial, bacterial, and fungal cultures. The material is also sent for rapid nucleic acid amplification tests such as Xpert MTB/Rif.[52]

Practice-point: Material should be sent for microbiological investigations (bacterial cultures, fungal cultures, mycobacterial cultures, and Xpert MTB/Rif) in sterile containers containing normal saline.[evidence level, UPP]

1.6 What are the immunohistochemistry and molecular tests that can be performed for diagnosing lung cancer in samples obtained by EBUS-TBNA?

With recent advancements in molecular profiling and targeted chemotherapy in lung cancer, additional material is required to perform ancillary tests. For subtyping the lung cancer, IHC can be performed on either the cell blocks or the slides. The commonly used IHC stains are thyroid transcription factor 1 (TTF-1) or napsin A (for adenocarcinoma), P63, or P40 (for squamous cell carcinoma), CD56 and chromogranin (for small cell carcinoma), and synaptophysin (for large neuroendocrine carcinoma).[53] The American Society of Clinical Oncology recommends performing molecular testing in advanced nonsmall cell lung cancer. The common molecular alterations to be tested include epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) translocation, ROS-1 mutation, and BRAF mutation.[43,44,53,54,55,56,57,58] To perform EGFR or KRAS mutation analysis, a minimum of 300 tumor cells are needed,[54] whereas for studying the ALK and ROS1 rearrangement, at least 100 tumor cells are required.[59] In those without the common molecular alterations, programmed cell death ligand-1 (PD-L1) expression should also be evaluated. Most of these molecular tests can be performed on the samples obtained by EBUS-TBNA. The molecular analysis can be performed on the cell block or scraping the slides after destaining.[60,61] The minimum number of cells required to perform these tests depends on the method used to perform the molecular analysis.[57,62] At least 40% of tumor cells should be present to perform real-time polymerase chain reaction.[54] For performing next-generation sequencing, the specimen should have at least 50% tumor cells.[43,62] However, a recent molecular testing guideline for selecting patients with nonsmall cell lung cancer for treatment with targeted tyrosine kinase inhibitors suggested that mutation may be detected in a sample with as little as 20% cancer cells.[58] A minimum of 100 viable tumor cells are required on the stained slide from the cell block to be tested for PD-L1[44,63]

Practice-points:

• A panel of IHC should be performed to subclassify the type of lung cancer if the morphological appearance does not result in a conclusive diagnosis.[evidence level, I]

• The samples obtained by EBUS-TBNA should be used for genotyping the nonsmall cell lung cancer or looking for PD-L1 expression, where needed.[evidence level, I]

1.7 Should rapid on-site cytological evaluation (ROSE) be performed routinely?

ROSE is on-site evaluation of specimens obtained during EBUS-TBNA.[13,64,65] ROSE is useful as it provides quick feedback for sample adequacy and thus may enhance the procedural yield. It guides the operator to stop further sampling, change the site of sampling, direct the need for additional material for immunocytochemistry, and has the theoretical advantage of reducing the procedure time.[9,13,18,66] However, it adds to the cost of the procedure (need for a cytopathologist) and depends on the availability of a cytopathologist.[67] A recent meta-analysis of RCTs studying the impact of ROSE on the diagnostic yield of EBUS-TBNA found no difference in the diagnostic yield of EBUS-TBNA, with or without ROSE.[13] The use of ROSE during EBUS-TBNA, however, did reduce the number of needle passes. The use of ROSE also reduced the need for additional bronchoscopic procedures required for making a final diagnosis.

In practice, the use of ROSE during EBUS-TBNA is especially beneficial in patients with co-morbid illness and those with sarcoidosis, where it reduces the need for additional procedures to make a diagnosis.[13] It also has utility in lung cancer staging because once a N3 node is positive for malignant cells, further sampling of other lymph nodes is not required, thereby considerably reducing the procedure time.[13] Finally, ROSE is helpful in patients with lung cancer to determine the adequacy of the aspirate for molecular testing.[68]

Practice-point: Rapid onsite evaluation should be used during EBUS-TBNA, wherever available.[evidence level, I]

1.7.1 Which stain should be used to perform ROSE?

The commonly used stains for ROSE include Diff-Quik, toluidine blue, rapid Giemsa stain, May–Grunwald–Giemsa, and rapid hematoxylin and eosin (H and E) stain.[66,69] The choice of stain depends on the availability and the user preference as the type of stain does not affect the quality of the slide or the reporting.[39] Diff-Quik is the most commonly employed stain worldwide.[66] This stain highlights the cytoplasmic characteristics and background details like mucus, necrotic debris, or connective tissue stroma.[4] Some institutes use the rapid H and E stain to perform ROSE. Rapid H and E stain provides better nuclear details; however, it involves multiple steps and takes about 5 min to perform ROSE.[69] Toluidine blue stain is another commonly available solution that can be prepared by dissolving 0.5 g of crystalline toluidine blue in 20 ml of 95% ethanol. The solution is diluted with 100 ml of distilled water, filtered, and refrigerated. For ROSE, air-dried smear is immersed in Coplin jar containing toluidine blue stain for 45–60 s and then washed with water. Alternatively, the stain can be poured over the air-dried slide and washed after a minute.

Practice-points:

• Wherever available, Diff-Quik may be preferred to perform ROSE of EBUS-TBNA samples.[evidence level, II]

• Other stains such as toluidine blue and rapid Giemsa stains may be used to perform ROSE due to the ease of availability.[evidence level, UPP]

1.7.2 Who should perform ROSE?

Despite the theoretical benefit of ROSE, not many centers can routinely incorporate ROSE during EBUS-TBNA. Underuse of ROSE is mainly due to the lack of availability of a cytopathologist. ROSE can also be performed by using telecytopathology, a cytotechnician, a pulmonologist, or cytology trainee or fellow.[64,65,67,69,70] In a feasibility study, the overall concordance between the preliminary diagnosis and the final diagnosis was 96% for telecytopathology, and 93% for conventional cytopathologist or cytotechnician performed ROSE.[70] In another study, the efficiency of cytopathologist was increased by using telecytopathology.[71] The telecytopathology may be a substitute for on-site assessment of EBUS-TBNA using a digital camera attached to the microscope to transmit stained slide images via a secure Internet connection to a cytopathologist who can perform ROSE remotely, with the results communicated by a voice communication system to the proceduralist. However, it still requires resources including procurement of the system required to perform telecytopathology, availability of internet, training of personnel to stain slides, and transmit images, and, most importantly, the availability of a cytopathologist to interpret the images. A study demonstrated a very good (ĸ =0.91) correlation between a cytotechnician and a cytopathologist, who were both blinded to the final diagnosis.[67] Another study demonstrated substantial (ĸ =0.75) agreement between cytopathologists and pulmonologists. The agreement was excellent (ĸ =0.81) for malignant disease.[72] There was no difference in the diagnostic accuracy of samples between a cytopathologist and a pulmonologist.[72] A similar level of correlation and agreement was also seen between the cytopathologist and pulmonologist in reporting TBNA samples.[65] At many academic centers, the ROSE is performed by cytopathology trainees or fellows. In a study, the cytology trainees, as well as cytotechnicians, performed similarly in the interpretation of cytology specimens obtained by endoscopic ultrasound (EUS)-guided FNA.[73] However, the interpretation by both the trainees and the cytotechnicians is generally suboptimal compared to the attending cytopathologist.[73,74] It is likely that better training may improve the diagnostic accuracy of trainees and the cytotechnicians.

Practice-points:

• ROSE should preferably be performed by a cytopathologist.[evidence level, I]

• ROSE may be performed using telecytology, depending on the availability of resources.[evidence level, II]

• ROSE may also be performed by a cytotechnician (evidence level, II) or a cytology trainee (fellow) or a pulmonologist trained in the interpretation of cytology.[evidence level, III]

1.7.3 What is the training required to perform ROSE by noncytopathologists?

Unlike interpretation of chest radiology, which is an integral part of training, the training and interpretation of cytology samples is not part of the teaching curriculum during pulmonology training. There is a need to incorporate structured training in cytology in pulmonology programs to reduce the burden and overdependence on the cytopathologist for performing ROSE. In a previous study, a substantial correlation was found between a cytopathologist and a trained pulmonologist.[72] The pulmonologist underwent structured training in cytology for three months (18 three-hour sessions). During the three-month training period, the pulmonologist also acquired theoretical knowledge of pulmonary cytopathology by reading textbooks. In another study, a good correlation was demonstrated between a cytopathologist and a pulmonologist despite no formal training of the pulmonologist in cytology.[65]

Practice-points:

• Pulmonologists require training in slide preparation, staining, and interpretation of the cytology slides.[evidence level, UPP]

• A structured program involving didactic lectures and interpretation of cytology slides may be incorporated in the training curriculum of pulmonology at academic centers.[evidence level, UPP]

2. ROLE OF LIQUID-BASED CYTOLOGY (LBC) DURING EBUS-TBNA

2.1 What is LBC? How is it performed? Does it offer any advantage over conventional sampling?

Liquid-based cytology is an alternative technique of processing cytology samples. It provides a uniform cellular spread in a thin layer without overlap of cells.[75,76] This involves the direct placement of the sample in an alcohol-based liquid medium and processed in an automated LBC machine.[77] There are two FDA-approved LBC equipment, namely, the ThinPrep® (Hologic Inc., Bedford Mass., USA) and SurePath® (Tripath PREP, BD SurePath Inc., Burlington, N.C., USA). The sample obtained by EBUS-TBNA is directly transferred to a fixative (CytoLyt, Hologic Inc, MA, USA for ThinPrep; SurePath® preservative fluid, BD Tripath, Burlington NC, USA for SurePath).[75,78] In the case of a bloody sample, additional Cytolyt or SurePath solution is added until the sample is clear. The sample is then centrifuged at 600 g (for 10 min) or 1300 g (for 5 min).[75,79] The supernatant is discarded, and the material is transferred to a vial containing cytopreservative solution (PreservCyt; Cytcy, Co [ThinPrep]) and allowed to stand for 15 min to enable fixing of the cells. The slides are then prepared with the help of T2000 ThinPrep system that prepares and stains the slides automatically. For SurePath, the sample is centrifuged at 2000 g for 10 min.[78] The supernatant is discarded, and the pellet is resuspended in 4 mL of separation reagent (PrepStain Density Reagent; BD Diagnostics).[78,80] The suspension is again centrifuged at 600 g for 5 min, and the supernatant is removed, and the pellet is transferred to the automated slide processor (PrepStain Slide Processor), in which the slides are automatically prepared and stained.[78,80] In addition, the remaining sample from LBC can be used to make cell blocks and perform ancillary tests such as FISH, flow cytometry, and other molecular tests.[43,44,79]

In a previous study, the use of LBC in EBUS-TBNA aspirates provided good quality specimens that were free from blood and air-drying artifacts.[77] Another study demonstrated a similar diagnostic yield of LBC or conventional smear method in samples obtained by TBNA.[76] In yet another study, the diagnostic yield of LBC (82.1%) was significantly higher than that of conventional smear (56%) method for mediastinal lymph node sampling using EBUS-TBNA.[80] However, the diagnostic yield was comparable to the conventional method in a recent study evaluating the role of LBC in the diagnosis of lung cancer.[75] Thus, more evidence is needed before LBC can be used routinely.

Practice-points:

• The use of LBC for cytological preparation during EBUS-TBNA provides a good quality specimen.[evidence level, II]

• LBC may be used for the processing of cytology specimens obtained by EBUS-TBNA, wherever available.[evidence level, II]

3. REPORTING OF CYTOLOGY SPECIMENS OBTAINED DURING EBUS-TBNA

3.1 What are the components of reporting?

Separate reporting should be done for the slides and the cell block.[81] The reporting of the EBUS-TBNA sample should have two components, including the adequacy of the specimen and the diagnostic category.[38,82] The report should also mention the use of immunocytochemistry or other ancillary techniques if any. A few criteria have been proposed for reporting of on-site evaluation of samples obtained by EBUS-TBNA [Table 2].[64,69,83,84,85]

Table 2.

Adequacy criteria for rapid-onsite cytologic evaluation

For cytology

>40 lymphocytes/hpf or the presence of germinal center or anthracotic pigment-laden macrophages[83]

>5 fields with at least 100 lymphocytes per low power field (×100) in a smear plus <2 groups of bronchial cells per low-power field (×100) or presence of germinal fragments[85]

Germinal center fragments, >5 fields at×100 magnification with at least 100 lymphocytes per field and <2 groups of contaminating bronchial cells per field[64]

A sequential approach comprising four criteria (core size ≥2 cm, presence of malignant cells, presence of microscopic anthracotic pigments, and mean lymphocyte density ≥40 cells/10 fields (×40)[84]

For molecular testing

A smear with greater than 40% for real-time polymerase chain reaction[54] or 50% tumor cells for NGS[43,62] or ≥ 20% tumor cells[58] for detecting mutation for use of tyrosine kinase inhibitors are required

A minimum of 100 viable tumor cells are required on the stained slide from the cell block. to be tested for PD-L1[44,63]

3.2 What is an adequate cytology specimen?

There are different criteria that are used to label a sample as adequate for routine cytology and for molecular testing.[81] However, these criteria are not uniformly accepted and require further validation. A sample is defined as adequate for evaluation in the presence of any of the following: germinal center fragments (centroblasts, histiocytes) or at least 100 lymphocytes per field (evaluated in at least five fields at × 100 magnification) with <2 groups of contaminating bronchial cells per field.[64] A sample is also labeled as adequate if it shows a definite diagnosis (malignancy or granuloma) irrespective of the number of lymphocytes. For molecular testing, a sample with as little as 20% of cancer cells may be adequate.[58]

3.3 What is a diagnostic cytology specimen?

A sample is labeled as diagnostic if it provides a definitive diagnosis such as malignancy, tuberculosis, sarcoidosis, and others. For uniformity of reporting, the diagnostic category should be reported as nondiagnostic, negative for malignancy (include granulomatous inflammation with or without necrosis and other), atypical, suspicious of malignancy or positive for malignancy (small cell cancer, adenocarcinoma, squamous cell carcinoma, and others).[86]

3.4 What are the essential items that must be described in every report?

The cytology report should always mention the adequacy of the sample, the diagnostic category, and the use of ancillary techniques, namely, immunocytochemistry, flow cytometry, and others [Table 3].[81]

Table 3.

A suggested template for cytological reporting of lymph node aspirate obtained using endobronchial ultrasound-guided transbronchial needle aspirate

Name Patient ID	Age Date of procedure	Gender Provisional clinical diagnosis
Cytology report
Adequate
For reporting
Germinal center		Yes/No
at least 100 lymphocytes per field at×100 magnification		Yes/No
<2 groups of contaminating bronchial cells per field		Yes/No
For molecular testing
>100 tumor cells		Yes/No
Proportion of tumor cells ≥20%		Yes/No
Inadequate
Diagnostic category
Diagnostic
Granulomatous inflammation (with or without necrosis)
Atypical
Suspicious of malignancy
Malignancy (small cell cancer, adenocarcinoma, squamous cell carcinoma or others)
Others (specify)
Nondiagnostic
Ancillary methods used		Report
Cell block
LBC
Immunocytochemistry
Flow cytometry
FISH
Special stains
Others (cell scraping)
Molecular testing used
Descriptive report
Final diagnosis

Practice-points:

• A structured template should be used for reporting cytology samples obtained by EBUS-TBNA.[evidence level, UPP]

• The cytology report should mention about the adequacy of the sample and the diagnostic category.[evidence level, UPP]

• The cytology report should also contain the details of the ancillary methods used (immunocytochemistry, flow cytometry, and others).[evidence level, UPP]

PRACTICAL APPROACH IN THE ENDOSCOPY SUITE

In all patients, we suggest that at least one air-dried and one wet-fixed slide be made from each lymph node pass [Figure 1]. The remaining material should be transferred to saline for microbiological analysis and 10% formalin for cell block preparation. The air-dried slide can be rapidly stained using Diff-Quik (or other rapid stains) for on-site cytological evaluation. If lymphoma is a diagnostic consideration, we suggest that the EBUS aspirate be transferred to RPMI solution, although some centers utilize normal saline for this purpose. An important factor in patients with lung cancer is to detect the tumor mutations, to enable personalized therapy accurately. In this context, 1 or 2 extra passes are obtained for bio-banking, which can then be used in the future [Figure 1].

Figure 1.

Suggested approach for specimen processing of aspirate obtained during endobronchial ultrasound-guided transbronchial needle aspiration

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.