EBUS-TBNA in the management of lung cancer:

NSCLC : A pathologist’s perspective

CME Kochi 2019

Dr S V Kane
Consultant Oncopathologist
Nanavati Superspeciality Hospital
Ex-HOD –Pathology-TMH
Mumbai
drsvkane@gmail.com
 Why should we focus on Indian scenario
Respiratory cytology ???

Worldwide, Lung cancer accounts

for 13% of all new cancer cases and
Incidence Mortality
19% of cancer related deaths

In India, lung cancer constitutes 6.9% Male 2nd most Most

of all new cancer cases and 9.3% of common common
all cancer related deaths in both Female 6th most 6th most
common common
sexes. Incidence of adenocarcinoma
Overall 4th most 3rd most
is increasing in INDIA common common

Study of Respiratory cytology

specimens have vital diagnostic role
in suspected primary & metastatic
lung cancer
Lancet Oncol 2014; 15: e205–12
What is necessary for optimal management of lung cancer?

• Accurate histologic diagnosis : Primary ca :Adenocarcinoma,

Squamous ca
• Accurate staging by pathologic evaluation

• Molecular analysis : EGFR, Alk , ROS , Met etc for targeted therapy

• Immunomarkers : PD-L1 for immunotherapy

Role of Respiratory cytology in general

Major role
• Diagnosis of malignant neoplasms involving lung
• both primary and metastatic
Minor role:
• Diagnosis of Opportunistic infections
• Specific inflammatory process
• Diffuse lung parenchymal disease
• Benign neoplasms,
Crucial role
- Availability of Molecular testing on cytology specimens &
targeted therapy have changed the scenario of diagnostic
cytology
- Staging of lung cancer by EUS-FNA
Importance of LN staging in lung cancer :
To operate or not : dilemma, planning treatment
Mediastinal
lymph node

Positive for Negative for metastasis/

metastasis reactive/Granulomatous

stage III
lung cancer Stage II

Inoperable- chemoth
/radioth/targeted therapy Surgery treatment
treatment of choice of choice
Mediastinal nodes

-Too many node

stations
- Difficult Approach
- Proximity to vital
structures
- Nodes may be small
yet positive
- Nodes may be
enlarged , yet
negative
- Radiologic evaluation

Pathologic evaluation
necessary
 EBUS – TBNA : New concept

• Endobronchial ultrasound guided transbronchial needle aspiration : a novel

technique , can be performed at the same time as flexible bronchoscopy
• to target the mediastinal lymph nodes at different stations as localized by US
• Minimally invasive procedure using real - time guidance. Which permits the
sampling of nodes that are smaller than 10 mm in short axis and near major blood
vessels
• Many Indications : Main indication is pathological nodal staging for lung cancer
- NSCLC
• Elaborate set up, proper training , space, team work
• Cytopathologists play crucial role.
 Indications for EBUS-TBNA

• Staging the mediastinal nodes in suspected non-small cell lung cancer (NSCLC),
• For diagnosis of both NSCLC and SCLC when there is no endoluminal tumor at
bronchoscopy [avoids the need for either a CT guided lung biopsy or mediastinoscopy].
• Suspected metastasis to mediastinum from known cancer…… common
• Mediastinal lymphadenopathy- unknown etiology
Sarcoidosis, Haematolymphoid malignancies, metastatic ca
• Suspected granulomatous disease -------common
• Sampling parenchymal pulmonary nodules
• Sampling endobronchial or peribronchial lesions
• Sampling mediastinal masses [ Other than lymphoid]
• As a research tool for tissue banking samples for later studies.
EBUS: seeing is believing, ….
Targeting occurs under Direct vision with Improved angulation & stylet
Good cellular yield is ensured
EBS Needle Appearance IHC FISH Molecular Flow
Technique gauge Test [ NGS] Cytometry
EBUS-FNA 22 Haemorrhagic Cell block/ + + +
Aspirate Smears

Biopsy 19 Worm like P block + + -

Thicker tissue
enmeshed in
blood

Special stains- mucin

IHC
Cell Block FISH
Molecular ( PCR/NGS)
[ Not Flow & Cytogenetics ]
 TBNA smear : Thin vs thick needle : microbx

22 G Needle 19 G

Courtesy Dr Moonim
EBUS Procedure

Busy room
Full of gadgets
and staff
Coordination is
the key for success
PET / CT Scan helps to target the most suspicious node for TBNA

Lower Paratracheal Nodes

 Procedure of ROSE
Non guided or guided FNAC and biopsy procedure

Fix Smears in FNAC / Biopsy cores

EtherAlcohol from same location for
- 30 seconds routine staining
Stain with 1%
T Blue –
10 Seconds Adequate and
Mount In representative
Glycerine

Repeat and
Non-
recheck
representative
Set Up Required for Rapid Staining of smears: ROSE
AR = Adequate and Representative

NR = Not representative/non diagnostic

Non lesional cells: bronchial epithelialcells
Cellular and lesional cells but trapped in
clot, Lesional cells seen but scanty

Overall it is cost- effective, time effective, and

simple technique which immensely improves
the patient care

Rose has become a key component of EBUS-TBNA

 EBUS-TBNA : Representative samples = AR

Reactive node
Neg for
metastasis

Node positive for

metastasis
NSCLC – Adenoca

NSCLC– PD
Carcinoma
Non-representative samples = NR
1 Non-lesional cells
Bronchial cells
macrophages
2 Obscuring artefacts
• Haemorrhage
• Necrosis
• Native / stromal cells
Drying artefacts
3 Thick tissue core
like material
What do we achieve with ROSE
Avoid Repeat procedure
• Delay in dx
• Cost
• Morbidity/ Complication
• Patient compliance

Adequacy rate
• Trained cyto-technician
• Cytopathologist
• Operational cost
• Time consumed
• False neg
Role in primary diagnosis Cell block
65 /F, Central lung mass : 4cm in dia

TTF 1

Diagnosis : primary
pulmonary adenoca
 P63

71 / M referred from outside for

EBUS-TBNA: FNA from Subcarinal
lymph nodes.
Diagnosis – metastasis of PD carcinoma,
ICC Done on destained smear
P63 positive , TTF1 : NEG
DX = PD NK SCC
 Trouble shooting in EBUS - FNAC
Problems Solutions
Low cellularity, non-representative On site adequacy assessment : ROSE
sample, Necrotic material Trained cytotech / cytpathologist
3 passes
Air drying , delayed fixation/ crushed Adequate training , proper work instructions,
smear… technical issues assistance by cytotech & audit

Thick / Hemorrhagic/ aspirate , Training for quick jabs/ passes, less dwell time in
Cellular details obscured by Large the lesion, Internal stylet should be advanced and
sheets of bronchial columnar cells, withdrawn repeatedly to expel debris and blood
Blood, & Inflammation clots away [ operator dependent ]

Difficulty in the subtyping of NSCLC on Extra smears for ICC, cell block
morphology or benign from malignant
 Logistics about number of passes at EBUS
• 3 passes per station of lymph node [N2/N3 ]

• No 1 Pass : 3 smears stained by T blue for adequacy, If reqd ,more smears

• If adequate, smears go for restaining by PAP & Giemsa
• No 2 pass taken[ optional ] only if IHC or molecular testing reqd on nodal
sample as per evaluation
• No 2 pass mandatory if no 1 does not yield cellular & representative material,
6 smears , 3 for judging adequacy, If adequate , procedure stopped
• No 3 pass mandatory if first 2 passes do not yield cellular material
• Procedure abandoned if no material on smears after 3 passes
 Suboptimal thick smear
Large sheets of bronchial cells : Ask for second pass
Subcarinal LN in a
known C/O ca lung
PET/SCAN active =
consistent with
metastatic carcinoma
NSCLC-NOS, ideally
calls for second pass
 Recent case: 56/m, Bx proven NSCLC -
ON EBUS—TBNA : Suspect adenocarcinoma
ON review FNA dx: reactive bronchial cells ;

False positive results are uncommon and occur when

needle contamination by reactive bronchial columnar cells
or compact epithelioid granulomas are misinterpreted as
adenocarcinoma cells.

False negative results are encountered due to sampling

issues mainly in :
1. Patients with Micrometastasis in the mediastinal nodes.
2. Patients with occult nodal disease "outside" the reach
of EBUS.
3 scanty cellularity of dissociated carcinoma cells

False positive occurs rarely in EBUS Cytologic criteria of malignancy are different
 Our Learning curve
• Technical difficulties: sampling and smearing issues
• Too many / thick sheets of benign bronchial columnar cells
• Thick Haemorrhagic aspirate with scanty cells enmeshed in clot
• Anesthetic Jelly material
• Once in a while aspirate with too many anthracotic pigment laiden cells

Interpretative issues
• Reactive bronchial cells or adenoca
• Atypical cells ? Nature…. epithelioid cells or tumour cells
• Necrosis with few tumour cells or necrotizing granuloma
• Reactive lymphoid aspirate vs HLM vs small cell ca
EBUS-FNA: from Subcarinal lymph nodes
Bronchial cells
Material : Non-representative
First pass
Subcarinal nodes –
second pass
lymphocytes and
adipocytes :
negative for
metastasis

ROSE : AR-
Paratracheal nodes : first pass

Hypocellular, Occasional cluster

: Labelled Atypical cells [ AUS]

Actually Inadequate smear

Second pass needed.
 Metastatic
Adenocarcinoma
Second pass

ROSE = AR
Confirmed on Pap : Clusters of adenocarcinoma cells :
common sample in EBUS
 FNAC of the Mediastinal mass 50 /m : EBUS - TBNA

Pap 4X

CD 3 CK

Cellular aspirate showing biphasic cell

population , reactive lymphiod cells
admixed with bland epithelial cells
Algorithm for sequential passes for EBUS
Mediastinal
lymph node 3 smears stained by T blue for adequacy, If reqd ,more smears
If adequate, smears go for restaining by PAP & Giemsa
1st pass

Negative
Positive
2nd pass

End of Positive Negative

procedure
End of 3rd pass
procedure
Positive Negative
Additional passes
can be taken to End of End of
obtain material for procedure procedure
ancillary techniques
Procedure abandoned if no material on smears after 3 passes
 How to reduce NR ; teamwork
• Multiple passes to get good yield ………operator dependent
• Quick entry & withdrawal of needle – less dwell time- less blood

• Proper smearing technique ………cytotech dependent

• Immediate Fixation

• Training of cytotechnologist & pathologist regarding adequacy criteria…….. Cytopath

dependent
• Clinical and radiological correlation…. Team work
• Constant feed back from cytopathologists & regular audit
 ROSE : EBUS -TBNA FNAC diagnosis (n= 84)

Positive for malignancy : 18 Squamous carcinoma 05

Adenocarcinoma 13

Negative for malignancy : 64 Reactive Lymphoid cells 54

Granulomatous lymphadenitis 10

Atypical cells – can not categorize Adenoca [ false neg] 01

further
Non- representative on ROSE : NR 01
final DX negative for metastasis
 ROSE-EBUS@TMH: Total samples 84

Positive Negative
Atypical cells

True positive-18 True negative- 64

False negative-1 False positive- 0, 1 NR

Total positive - 19 Total negative - 65

Total 84
ROSE-EBUS statistics
• EBUS with ROSE can diagnose metastatic lung cancers with a sensitivity and
positive predictive value of greater than 90%.*
• Arch Pathol Lab Med. 2018;142:253–262; doi: 10.5858/arpa.2017-0114-SA

• ROSE-EBUS @ TMH for diagnosis of malignancy

• Sensitivity: 94.7%
• Specificity: 100%
• PPV: 100%
• NPV: 98.5%

• Secret of Success : dedicated team , EBUS for staging purpose ,Trained staff
Petals of ROSE in EBUS-FNAC

• Highly specific
• Reduces the number of cytology slides examined
• Avoids delay in diagnosis, also fatigue
• Ability to better triage the sample for ancillary studies →saves both
time and money
• Reduce complications →lower number of repeat procedures for
ancillary studies.
• Cost- effective, Saves time &improves patient care
Algorithmic flow of ROSE for EBUS -TBNA
 Ancillary techniques : EBUS -TBNA

• ICC Sample type

• FISH - Unstained smears
• MOLECULAR TECHNIQUES: - LBC
• Flow cytometry - Cell block
• Cytogenetics - Special medium
• NGS

• 3 slides of cytology, where in diagnosis of malignancy has

been confirmed
• Minimum 300 to 500 atypical cells which are true
representative for the diagnosis in each slide

The factor which makes needle bx superior technique, can be applied to cytology
 TRIAGING SAVES
TIME & COST FNA

Intra-procedural assessment / ROSE

• Abcess • Metastatic carcinoma • Reactive Lymphnode

• Granulomas • Hodgkin Lymphoma • Non-Hodgkin Lymphoma

Needle washings FNA– same / additional pass

• Microbiology • Cell block & IHC • Flow cytometry,

• Molecular Cytogenetics, Molecular
for adenoca • Cell block
Shared by Dr. Moonim
Alk : D5F3 Ab positive indicates Alk gene rearrangement
 Merits of EBUS – TBNA

• Superior sensitivity: 84-95% and specificity : 100%

[Cell yield better in > 10 mm nodes ]
• Easy access to a wide range of MEDIASTINAL nodal stations
• Real-time sampling
• Direct visualization of node
• Minimally invasive,
• Less risk of major vessel puncture / complications
• Shorter procedure time & Cost saving (as an out patient procedure) than
mediastinoscopy

* Medford AR, Bennett JA, Free CM et al. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-
TBNA): applications in chest disease. Respirology 2010;15:71–9. doi: 10.1111/j.1440-1843.2009.01652.x [PubMed]
Mediastinal Nodal staging

Yasufuku K, Nakajima T, Motoori K, et al.

Chest 2006;130(3):710–8
 Mediastinoscopy
was…. standard of care

Major Limitation
• Limited access to lymph node stations (ex subcarinal
station 7)
• Requires general anesthesia , Skills
• 2% morbidity & 0.08% mortality risk
•Not time and cost effective

Alternatives:
PET /CT or CT : False negative in smaller nodes [ <1cm]
and positives in granulomatous lesions, however do allow
targeting of the metastatic node. Sensitivities of CT VS PET
VS EBUS : 76.9%, 80.0%, and 92.3%, respectively;
Specificities were 55.3 %, 70.1%, and 100%, Conventional
TBNA: sampling without direct vision : NR samples
 Limitations of EBUS –TBNA

• Operator dependent.
• High initial capital costs – not afforded by all centres
• Dedicated staff and Organised activity required
• EBUS cannot image or sample subaortic and paraesophageal lymph nodes. [ 5,6,8,9 node stations]

• Potential for false negative in micrometastasis, Unaccessible nodes [sampling issues]

Complications reported are

• Bleeding requiring intervention
• Pneumothorax ,Sustained hypoxia
Affordability and tech expertise is the main issue. Its availability is institution-specific.
All cases which were negative on TBNA underwent mediastinoscopy & bx , only 3% cases showed positive nodes

Best suited for ……. Isolated hilar or mediastinal nodes … is becoming the standard of care for staging NSCLC
Indications of EUS –FNA
• Suspicious Subcarinal / paraoesophageal nodes , [station 7& 8] not
accessible by TBNA
• Poor Gen Conditions
• ALREADY endotracheal tube inside patient
• 85% - 3 PASSES required , [ if no ROSE done]
• Team work : Pulmonologist & GI surgeon
 Towards increasing efficiency
Be aware of what is expected -
• Look for host cells first , then lesional cells
• Take extra care in handling scant material, may be necrotic
• Malignant Cases: Usually fast, few passes, cellular yield
• Negative Cases: Require more passes, radiologic corelation
• MIN Dwell time to avoid clotting like surgical strike

Beware of diagnostic Pitfalls

• Benign neoplasms mimicking malignancy
• Reactive atypia vs. Tumor
• Needle Contaminants
• More than one entity : tumour & granuloma
Data collection
and entry imp
 Take home message
• pre-operative Nodal staging is mandatory in the management of
NSCLC.
• ROSE →Essential part of EBUS-FNAC procedure for improving
diagnostic yield & Prompt triaging of sample for ancillary techniques
• Correlation with clinico-radiological findings, ROSE & ancillary
techniques will help in improving the diagnostic accuracy.
• Cytology with cell block offers a final diagnosis with Ancillary
techniques where biopsy is not feasible or has failed.
• EBUS -TBNA with ROSE is an ideal technique recommended for
accurate nodal staging in NSCLC.
• Best example of team work
 EBUS Team for Improving Patient Care

Clinicians
Team EBUS@TMH

Anesthesiologist
Radiologist
Thoracic surgeon
Pulmonologist
Radiologist
Cytopathologist Cytotechnician
Support staff

Best example of team work Pathologist

Shared by Dr. Moonim
THANK YOU

Clinicians

Pathologist Diagnosis Radiologist

Cytotechnologis
t
 Need for EBUS-TBNA for staging

Alternatives:
• CT, PET /CT : False negative in smaller nodes [ <1cm] and positives in
granulomatous lesions, however do allow targeting of the metastatic node.
• Conventional TBNA: sampling without direct vision : False negative, NR
• Mediastinoscopy and biopsy : gold standard
 ABC of EBUS –TBNA sample Evaluation

• A wareness & knowledge of adequacy & morphologic spectrum

• B eware of Pitfalls & Contamination

• Carefully analyze material & triage appropriately

Potential pitfalls in diagnosis of Lung cancer
Restrict dx of cancer

• No discreet mass on FOB or radiology

• Low cellularity
• Clean background
• pure inflammatory background with epithelioid cells
• Drying artifact
• Reactive bronchial cells with some atypical cells
• Overlapping morphology…….experience & expertise tested here

Int j clin exp pathol 2010; 3(4): 367–385.

Important terms in ROSE-FNA
• Representative
• Adequate
• AR = Adequate and Representative
• NR = Not representative/non diagnostic
• Non lesional cells: bronchial epithelial
• Cellular and lesional but trapped in clot
• Lesional cells see but scanty
• Cellular material
Multiple irregular cell
clusters
• Large atypical cells
• Adequate &
representative =A R
Diagnosis Frequency [ 71] Percent
Metastatic NSCLC 45 63.3
Tuberculosis 4 5.6
Infective/Inflammatory 8 11.2
Haemato- Lymphoid
1 1.4
malignancy
Metastatic carcinoma of varied
primary Sites
13 18%
SCLC 1 1.4
Ca Oesophagus : 4 5.6
Ca Breast 1 1.4
Ca Gall bladder 1 1.4
Ca ovary 1 1.4
Ca Bladder 1 1.4
Ca Colorectal 1 1.4
Head & Neck SCC 2 2.8
Adenoid Cystic Ca 1 1.4
 NSCLC- No Morphologic E/O differentiation

IHC – P40, CK 5, TTF 1, Napsin A

TTF 1 / Napsin A +ve P40 / CK 5, +ve All -ve

Adeno Ca (Soild) Non keratinising SCC NSCLC - NOS

EGFR, ALK, PIK3CA, CDKN2A, No Further

ROS1, MET TP53 Markers
 Conclusions of studies in different countries
• The diagnostic yield of mediastinal staging by EBUS-TBNA is high, with a reported sensitivity of 90% and a
specificity of 100%.
Diagnostic yield of endobronchial ultrasound-guided transbronchial needle aspiration for mediastinal staging
in lung cancer*
• Compared to CT and PET, EBUS-TBNA has a high sensitivity as well as specificity for mediastinal and hilar
lymph node staging in patients with lung cancer. EBUS-TBNA should be considered for evaluation of the
mediastinum early in the staging process of lung cancer.
• EBUS-TBNA is a safe and efficient method with high sensitivity
and specificity in the diagnosis of mediastinal and hilar lesions.
Uniquely combining with IHC and genetype has important clinical
value in subtype diagnosis and guiding the treatment strategy.
• Endobronchial ultrasound guided transbronchial needle aspiration combining with immunohistochemistry
and genotype in lung cancer:
• A single-center, 55 cases retrospective study
• Aiqun Liu a, b, Liwen Qian a, b, Yi Zhong b, Xiaoling Lu a, **, Yongxiang Zhao a, *
• Annals of medicine & surgery 2017; 23: 1-7
EBUS-TBNA 22 ROSE : Cytology
gauge needle preparation
Contraindications
Relative
• INR >1.4
• Current anti-platelet agents OR anticoagulant therapy (stop 1 week prior)
• Myocardial infarction (postpone > 6 weeks)
• Severe hypoxemia at rest
• Arrhythmias
• Suspected lymphoma (clinically inappropriate)
• Thrombocytopenia

No obsolute C I
Increasing efficiency: what is std cyto
procedure , unmet needs
• 65% positivity
• COST of instrument – 1 CRORE, Olympus Needle 30000 INR
• Low vol : ,150 procedures / yr for training
• No of passes: for sarcoidosis = 6passes , malignancy : less
• Gauge of the needle impact not known
• Passes of the needle per station 10 vs 20
• Rest everything in saline, cell block thromboplastin
• 3 passes ideal station 7/8 easy to aspirate
• Station 11/ 12 -difficult
Case 5

• Post Bronchoscopy Sputum X 3

• Male / 57
• C / O – puffiness of face, anorexia, chest pain
• CT: lesion in RUL with large mediastinal mass with involvement of SVC
• Bronchoscopy: Nil endobronchial
Thorns of ROSE in EBUS-FNAC
• Bronchial epithelial cells
• Anthracotic pigment & macrophages
• Squamous cells from upper aero-digestive tract
• Cost of the procedure
• Missing the lesion!!(3 of our cases)
Case 5
Case 5
Your Opinion?
ROSE for primary dx Second pass :
Atypia of undetermined significance PAP smear = adenocarcinoma
Calls for second pass
Case 5
• Post Bronchoscopy Sputum X 3
• Male / 57
• C / O – puffiness of face, anorexia, chest pain
• CT: lesion in RUL with large mediastinal mass with involvement of
SVC
• Bronchoscopy: Nil endobronchial
• Bronchial lavage – Neg
• Post Bronchoscopy Sputum X 3 – Scanty cells suspicious of
Adenocarcinoma
• Biopsy from Ant. Mediastinal mass – NHL
• FP – Interpretative error
• On retrospect these are reactive glandular cells – smooth
regular nuclear membrane , no anisonucleosis, chromatin is fine
 Team EBUS@TMH

• Anesthesiologist
• Radiologist
• Thoracic surgeon
• Pulmonologist
• Cytopathologist
• Support staff

Best example of team work

 Complications

 EBUS and EBUS-TBNA are usually safe procedures.

 No serious complications.
 TMJ dislocation.
 Reported complications are agitation, cough, hypoxia,
laryngeal injury, fever, bacteremia, bleeding,
pneumothorax, and broken equipment becoming stuck in the
airway.
 Mediastinal abscess has been reported as a case report.
 Complications related to upper airway local anesthesia are
laryngospasm, laryngeal edema, bronchospasm,
and cardiac arrhythmias.
 Complications attributable to procedural sedation are
respiratory depression, cardiovascular instability, vomiting,
and aspiration.
• EBUS bronchoscope - similar in dimensions to a standard adult
fiber optic bronchoscope.

• Linear continuous B-mode ultrasound image, with color

Doppler.

• Proximal to the US probe, and at 30 degrees to the long axis of

the bronchoscope is a biopsy channel.

• A disposable latex balloon placed over the US probe, which is

inflated with sterile water to provide a fluid interface between
the probe and the tracheal wall.
 Current Management of NSCLC
- Primary diagnosis on image guided core biopsy , bronchoscopic bx,
cytology is complementary to histology
- Adenoca, primary pulmonary origin, reflex molecular testing

- Early Mediastinal Nodal staging equally important in determining

outcome and planning treatment strategy including operability

- Options available for pretherapy Nodal staging

• Mediastinoscopy + bx…. Gold standard
• Video-assisted thoracoscopy & biopsy
• Conventional TBNA
• EUS with FNA
• EBUS-TBNA
Lymphocytes lost in the background :
AR- neg for metastasis
 Contraindications

- Similar to standard fiberoptic bronchoscopy procedure

- Relative CI

• INR >1.4
• Clopidogrel (stop 1 week prior)
• Myocardial infarction (postpone > 6 weeks)
• Severe hypoxemia at rest
• Arrhythmias
• Suspected lymphoma (clinically inappropriate)
• Similar to standard fiberoptic bronchoscopy procedure
Respiratory epithelial cells
Bronchial columnar
cells

In a well fixed and

processed smear cilia &
t plate easily identified
 Thick smear
– broncial
lavage

thin smear
bronchial
lavage

Superficial
squamous
cells
Reactive
columnar
cells
ROSE-EBUS@TMH for diagnosis of malignancy

• Sensitivity: 94.7%
• Specificity: 100%
• PPV: 100%
• NPV: 98.5%
Differentiation between malignancy and
benign pathologies
Cytologic Squamous Adenocarcinoma Squamous Pulmonary Bronchial cell Reactive bronchial
features carcinoma metaplasia infarction hyperplasia cells
Cellularity Usually Usually Normo to Hypocellular Hypercellular Usually
hypercellular hypercellular hypocellular Hypocellular
Pattern Cohesive sheet, 3D clusters, acinar, Single cells, sheets Tight clusters, #D Papillary clusters, Cohesive clusters
single cells, keratin single cells, loss of of cells clusters Polarity
pearl+/- polarity maintained
Cell type Strap cells, fiber Cuboidal, Mature and Cuboidal squamoid Columnar, cilia and Columnar , cilia
cells, polygonal cells, columnar, immature squamous terminal plate and terminal plate
tadpole cells polygonal, no cilia cells, spider cells present present OR absent
Cytoplasm Dense, variable in Scant to moderate Dense, basophilic, Scant Moderate, variable Moderate to
amount with fine to coarse usually more mucus producing abundant
vacuoles cells
Nuclear Irregular Irregular Regular Regular , may be Smooth Smooth
membrane irregular
Nuclear Variable chromatin, Fine granular with Variable chromatin, Coarse Fine granular Usually fine, with
chromatin very occasional prominent(>1) occasional nucleoli small nucleoli.
nucleoli nucleoli
N:C ratio Usually high, Variable, usually Variable with round High Low Low
anisonucleosis + high central nucleus

Background +/- Necrotic Necrotic +/- non necrotic Degenerative, Non-necrotic/ Non necrotic, may
Hemosiderin laden inflammatory be inflammatory
macrophages
Differentiation between malignancy and
benign pathologies
Cytologic features Small cell carcinoma Reserve cell Lymphoproliferative
hyperplasia disorder
Cellularity Hypercellular Variable Hypercellular
Background Necrotic Clean, inflammatory Non necrotic
LG body
Pattern Individual cells, Cohesive cluster Dyscohesive, uniform
Loose cluster, No moulding
Indian file
moulding
Cell type Neuroendocrine Cuboidal Lymphoid
pleomorphism
Cytoplasm Scant, easily stripped Scant, indistinct Scant, basophilic rim

Nuclear membrane Smooth Smooth Irregular, notched

Nuclear chromatin Stippled Fine granular Coarse
N:C ratio High High High
Background Necrotic Clean, inflammatory Non necrotic
LG body
Petals of ROSE in EBUS-FNAC
• Highly specific
• Effective in reducing additional procedures
• Cost effective
• Reduces the number of cytology slides examined
• Avoids delay in diagnosis
Petals of ROSE in EBUS-FNAC
• Ability to better triage the sample for ancillary studies →saves both
time and money
• Reduce complications →lower number of repeat procedures for
ancillary studies.
• Improved patient care
Thorns of ROSE in EBUS-FNAC
• Bronchial epithelial cells
• Anthracotic pigment & macrophages
• Squamous cells from upper aero-digestive tract
• Cost of the procedure
• Missing the lesion!!(3 of our cases)
 Ancillary techniques : nothing that one can not do
on FNA Sample

• Necessary to Improve diagnosis & evaluate prognosis

• ICC----- DX & typing of carcinoma

• FISH-ALK, ROS 1 & MET targeted therapy---
• Molecular diagnostics----- typing & prognosis,
• Flow cytometry------ Dx & typing of Lymphoma
• NGS / WES/ WGS

• ADD moofi & nitin data

Reasons for NR
• Material trapped in blood clot
• Obscuring inflammatory cells
• Air drying
• Inadequate sampling or insufficient specimens due to scant cellularity
• Tumors with cystic change
• Needle positioning outside the target tissue esp. in small lesions
• Marked necrosis and haemorrhage
• Predominance of stromal / native cells
Pathology: In 2015, the World Health Organization published the
fourth edition of the Classification of Tumours of the Lung, Pleura,
and Thymus which included several important changes: (1)
classification applied to small biopsy and cytologic samples, (2)
molecular testing for treatment selection, (3) inclusion of IHC
markers for more precise classification of NSCLC, (4) changes in
the classification of squamous carcinoma and adenocarcinoma,
and (5) new genomic information for various types of lung
cancers.
Procedure
• Procedure room: Endoscopy room or operating room.
• Anesthesia: Concious sedation or GA, GA preferred as controlled apnea is
beneficial for sampling smaller nodes (<10 mm). Laryngeal mask airway with iv
anaesthesia.
 At the very beginning…
• Novel technique is always looked upon with dread

• How are we going to take out time for the EBUS procedure?
• How much time a single procedure will consume???
• How many slides to prepare per pass???

• We learnt the lessons as we proceeded

• Limiting no of smears to be stained per pass to 2-3
• Additional material & second pass collected for cell block
• Cell block preparation – after confirming representative aspirate
• Used for ancillary technique – esp. IHC and special stains
Is it representative???

Thyroid FNAC

2nd attempt

Clinical and radiological

impression – suspicious for
malignancy….

Final diagnosis – Papillary thyroid carcinoma with cystic change

 Procedure of EBUS –TBNA

• Needle is inserted in the node under real-time US guidance.

• The stylet of the needle is left in place on the first puncture to
minimize bronchial cell contamination
• The target is stabbed 10-15 times without suction.
• The specimen is then air-flushed on a clean slide.
• The same procedure is repeated three times at every lymph node
station for adequate material. [3 needle passes per node station]
• Needle size: 22G
• Average time : 15min
• On site evaluation [ROSE] : reduces no. of passes.