Cytologic assessment of bronchopulmonary lesions

Malvika tripathiResident of pathology

Contents

1. Embryology of respiratory system,2. Anatomy of respiratory system,3. Histology of respiratory system,4. Cytologic sampling techniques,5. Cytology of respiratory tract,6. Bronchopulmonary lesions,7. Cytology of bronchopulmonary lesions.

Embryology

Anatomy

Histology of normal respiratory tract

• Two principal types of epithelium are encountered within the

upper respiratory tract and the bronchial tree:-

o nonkeratinizing, stratified squamous epithelium,

o characteristic respiratory epithelium.

• Within the trachea and the main bronchi, the epithelium is

truly stratified with two, three, or more layers of columnar cells.

• Smaller bronchial branches, lined by nonciliated columnar and

cuboidal cells which are single layered low columnar or

cuboidal epithelium.

• The terminal bronchiolar epithelium includes Clara cells,

nonmucus-secreting cells that produce surfactant .

• A small number of basally placed neuroepithelial cells known

as Feyrter or Kulchitsky cells also are present, primarily at

airway bifurcations.

• Alveoli –

• Ultra structural studies have shown the wall of the alveolus to

be surfaced by two types of epithelial cells, pneumocytes type I

(90%)and pneumocytes type II .

• Pneumocytes type I are flattened cells, few in number, with

extremely attenuated cytoplasm .

• The remaining 10% of the alveolar surface is occupied by more

plump, rounded, or cuboidal pneumocytes type II.

• Pneumocytes II are cytokeratin +ve.

Cytologic sampling methods

1. Sputum sample.

2. Bronchial brushing.

3. Bronchial aspirates and washings.

4. Bronchoalveolar lavage (BAL).

5. Needle aspiration biopsy.

a) Percutaneous aspiration biopsy.

b) Transbronchial aspiration via fiberoptic bronchoscopy.

Sputum sample

• By far the simplest and most useful method of investigating the

respiratory tract.

• Patients should be told to clear their nasal passages and rinse

their mouth with water, discarding that material before collecting

a specimen.

• Ideal diagnostic material is obtained from a spontaneous deep

cough, which should be expelled directly into a wide-mouth

container with fixative.

• Often the best specimens are obtained on arising in the morning.

• Sometimes the material submitted may consist entirely of

mouth contents or saliva that is of no diagnostic value.

• For patients with a nonproductive cough or no cough, it is

possible to induce coughing by inhalation of a heated aerosol

of 20% polypropylene glycol in hypertonic (10%) saline.

• One container may be used to collect three or four deep cough

specimens.

Processing of sputum

• Pick & Smear technique -

Appearance of sputum

• Bloody – Hemoptysis• Rusty – Pnemococcal lobar pneumonia• Bloody and gelatinous (Red current jelly) – Klebsiella

pneumonia• Green – Pseudomonas infection• Purulent and separating into 3 layers on standing –

Bronchiectasis,lung abscess.• Pink, frothy (air bubbles) – Pulmonary edema• Copious amounts of purulent sputum – Lung abscess,

bronchiectasis, bronchopleural fistula.

Bronchial brushings

• With the introduction of flexible bronchoscopes capable of

reaching sub segmental bronchi, the cytologic diagnosis of

lung cancer relies heavily on direct bronchial brushings.

• The method permits sampling of a visualized mucosal

abnormality or systematic sampling of all segmental bronchi

to confirm and localize occult in situ or early invasive

carcinomas detected by sputum cytology or suspected

radiologically.

• Cell samples are obtained with a small brush threaded

through a separate channel in the fiberoptic bronchoscope,

guided to a selected site under visual control.

• Brushings may be supplemented by tissue biopsies or by

transbronchial aspiration biopsy of lesions within reach of the

fiberoptic bronchoscope.

Bronchial aspirates and washings

• Bronchial washing specimens are obtained under

bronchoscopic guidance by first aspirating the accumulated

contents of the bronchus (or bronchi) in an initial sample.

• Then, additional samples are obtained by repeatedly instilling

and re aspirating (about 50 ml) normal saline from the

selected bronchus or bronchi.

Bronchoalveolar lavage (BAL)

• BAL was introduced initially as a therapeutic procedure to

clear the alveolar spaces of accumulated secretions blocking

gaseous exchange. E.g. – Bronchial asthma, alveolar

prteinosis.

• Subsequently, the technique has been used for diagnostic

purposes primarily in suspected Pneumocystis carinii

pneumonia, replacing open lung biopsy and in the diagnosis

of interstitial lung disease.

• Uses of BAL –

Procedure –

Under local anesthetic, the bronchoscope is passed to the lung

segment of interest, usually a secondary or tertiary bronchus,

and wedged to occlude the bronchial lumen.

From 100 to 300 ml of normal saline is instilled in 20 to 50 ml

aliquots, re aspirated, and the collected fluid is forwarded to

the laboratory for processing.

• Evaluation is based on differential cell counts and

immunophenotyping the cells present, as well as chemical

analysis and bacteriologic study of the fluid retrieved from the

alveolar spaces.

• If the lavage is properly performed, the cell content will be

limited to the epithelium of the bronchioles beyond the point

of occlusion and to the contents of the alveoli, mainly alveolar

macrophages and inflammatory cells.

Processing and lab assessment of BALProcess and analyze BAL promptly (e.g., cells in nutrient-poor media such as saline should be processed within 1 hour)

Avoid containers that promote cell adherence to container surfaces

Use nutrient-supplemented media for prolonged storage (e.g., 12 to 24 hours) if necessary (discard specimens obtained more than 24 hours prior to processing and analysis)

Keep cell suspensions at 4 degree centigrate if not analyzed immediately

Obtain nucleated cell counts via a hemocytometer and identify cell subpopulations via cytocentrifugation with staining

Perform analyses of BAL fluid and cells as needed to diagnose infection

Observe and report the following:1. Volume and gross appearance (color and turbidity) of uncentrifuged BAL fluid2. Absolute number of total nucleated cells and total number of red blood cells3. White blood cell differential percentages4. Percentage of epithelial cells that represent total nucleated cells5. Other specific findings (e.g., plasma cells, mast cells, foamy alveolar macrophages.

Needle aspiration biopsy

• There are two techniques of pulmonary aspiration biopsy –

1. Percutaneous aspiration.

2. Transbronchial aspiration via fiberoptic bronchoscopy.

• Computed tomography (CT) or, less commonly, ultrasound is

used to guide the direction and depth of insertion of the

biopsy needle; fluoroscopy is no longer used.

• Contraindications to Percutaneous needle biopsy include the

following:

1. Hemorrhagic diathesis

2. Anticoagulant therapy (unless previously discontinued with

restoration of normal clotting time)

3. Severe pulmonary hypertension

4. Advanced emphysema

5. Suspected arteriovenous malformation or aneurysm

6. Suspicion of hydatid cyst (see below)

7. Uncooperative patient

• When the lesion is close to the chest wall, it can be reached

with a thin, relatively short needle (external diameter, 0.6

mm; length, 10 cm).

• Thin needles may be unsuitable for small (2 cm or less) deep-

lying lesions. Such needles may bend during passage through

the pulmonary parenchyma, and the target may be missed.

• A wider bore, sturdy needle (0.9 to 1 mm external diameter)

will not bend easily and may be more accurately guided to the

lesion. A stylus inserted into the needle lends additional

rigidity to the needle and also prevents tissues from the

thoracic wall entering the lumen of the needle as it is

inserted.

• First suggested by Wang et al (1981),

• Used to sample enlarged para-hilar or para-bronchial lymph

nodes or other near- hilar masses that cannot easily be reached

by percutaneous needle biopsy.

• Performed during bronchoscopy when an extrabronchial lesion

is suspected.

• A thin, flexible needle is inserted through the bronchial wall into

the suspected lesion via the bronchoscope, and the cellular

material is aspirated and processed as for percutaneous biopsies.

Transbronchial aspiration via fiberoptic bronchoscopy

Cytology of respiratory tract

• Squamous epithelium –

• Similar in all respects to the superficial and intermediate

squamous cells of the female genital tract.

• There may be karyomegaly of occasional cells without

apparent significance .

• They may be present singly, but are often in plaques and

encountered more commonly in inflammatory disorders of

the oral cavity.

• Squamous pearls or anucleation can also occur.

• Respiratory epithelium –

• Cells derived from this epithelium are uncommon in sputum

and are typically seen in specimens obtained by bronchial

brushing or aspiration, or after other procedures that dislodge

them from their epithelial setting, such as bronchoscopy.

• If they are present at all in a sputum specimen, it is an

indication of prior instrumentation, trauma, or severe cough.

• Therefore, their presence in a specimen is not absolute

insurance of origin from the lower respiratory tract.

• Ciliated cells –

• Respiratory epithelium is readily recognized in cytologic

material by the presence of ciliated columnar cells.

• Columnar cells may appear singly or in groups or clusters of

cells, depending on how forcefully they have been dislodged.

• At the periphery of such clusters, normal ciliated cells may

appear at a right angle to the main axis of the cluster, giving

the impression of feathering.

• Size - about 30 to 50 µm in length and 10 to 15 µm in width.

• They are typically cilia bearing and columnar in configuration.

• Cytoplasm - homogeneous and lightly basophilic or less

commonly eosinophilic. Rarely, small mucus vacuoles may be

observed.

• Nuclei - very finely textured and oval in shape, with their long

axis corresponding to the long axis of the cell. Sometimes, the

nucleus appears to be larger than the transverse diameter of

cell, resulting in a slight bulge at the level of the nucleus.

Bronchial brushing specimen showing bronchial cells in a cluster with some cells projecting out of the cluster to give a “feathering” appearance.

Bronchial cell with a nuclear hole attributed to an artifact of preparation has no diagnostic significance.

• Alveolar macrophages –

• The alveolar macrophages are of great importance in

evaluating cytologic material from the respiratory tract.

• Macrophages are most abundant in sputum specimens from

cigarette smokers and in specimens from patients living in

dusty environment, for e.g. farmers.

• In BAL specimens, they are the predominant cell type, and

present in abundance.

• Size - 10 to 25 µm or more in diameter.

• Shape - spherical or oval .

• Cytoplasm - usually amphophilic, may be abundant or limited in

amount, basophilic or acidophilic, and usually contains a variable

amount of phagocytized gray, brown, or black granular dust

particles, hence the name dust cells.

• Nuclei - vary in size and number but are generally round, oval, or

kidney-shaped, about 5 to 10 µm in diameter, with fine, evenly

dispersed chromatin and small nucleoli.

• Cells with more than 10 nuclei in BAL specimens, most common

in sarcoidosis.

Alveolar macrophages

• Goblet cells –

• less common than ciliated cells.

• basally placed nucleus and distended supranuclear cytoplasm

that is tightly packed with faintly basophilic tiny vacuoles

representing packages of mucus.

• Leucocytes –

1. PMNs - very common in cytologic specimens from the

normal respiratory tract, especially in cigarette smokers.

However, a finding

of numerous PMNs,particularly in the presence of necrotic

material in an acutely ill patient, suggests a major

inflammatory process such as pneumonia or abscess.

2. Eosinophils - or the elongated Charcot-Leyden crystals

suggest an allergic process, such as bronchial asthma.

3. Lymphocytes - singly or in pools, are a common finding in

various inflammatory disorders.

• In the benign conditions, there is typically a mixture of mature

small and medium lymphocytes with scattered large reactive

lymphoblasts and phagocytic macrophages .

4. Monocytes - may be observed occasionally and are now

known to be precursors of the larger alveolar macrophages.

• Non cellular endogenous material

Curschmann's Spirals Mucus blobs / Inspissated mucusCorpora amylacea

Benign cellular abnormalities 1. Abnormalities of bronchial epithelium

E.g. Ciliocytophoria, Bronchial cell hyperplasia, Immobile cilia syndrome

2. Abnormalities of squamous epithelium E.g. Inflammatory changes, Pap cells.

3. Abnormalities of alveolar epithelium E.g. Hyperplasia of type II pneumocytes

4. Abnormalities of pulmonary macrophages E.g. Hemosiderin laden macrophages

Inflammatory changes

1. Acute bacterial inflammation E.g. Pneumonia, lung abscess, bronchitis

2. Chronic inflammatory processes E.g. Chronic bronchitis and Pneumonia

3. Specific inflammatory processes E.g. TB, sarcoidosis, Actinomycosis, nocardiosis

4. Viral Infections E.g. HSV, CMV, RSV, measles, adenovirus, parainfluenza

5. Fungi E.g. Cryptococcus, Blastomyces, Histoplasma , Aspergillus, Candida

6. Parasites E.g. Echinococcus, Giardia, Lung flukes

Other benign conditions

1. Alveolar proteinosis2. Malakoplakia3. Rheumatoid granuloma4. Gaucher disease5. Sclerosing hemangioma6. Follicular bronchitis

Tumors of lung

1. Squamous cell carcinoma - Well differentiated /Keratinizing - Poorly differentiated/ Non keratinizing

2. Large cell undifferentiated carcinoma3. Small cell undifferentiated carcinoma

- Oat cell - Intermediate cell type

4. Adenocarcinoma - Adenocarcinoma of central origin - Bronchoalveolar carcinoma

5. Adenosquamous carcinoma6. Mucoepidermoid carcinoma7. Spindle and giant cell carcinoma8. Neuroendocrine tumors

Tuberculosis

• Caused by mycobacterium tuberculosis.• The common pulmonary form is caused by inhalation,

whereas the rare intestinal form is caused by ingestion, usually in milk.

• The upper lobes are first involved, and as the disease progresses, large areas of confluent granulomas undergo caseous necrosis.

• Expulsion of the necrotic material through the bronchi leads to formation of cavities that are the hallmark of late stages of the disease.

• Epitheloid cells, Langhan’s giant cells and caseous necrosis are hallmarks of TB.

Langhans’ giant cellEpitheloid cell

Cluster of Epitheloid cells and spindle cells suggestive of granuloma

Sarcoidosis

• This disease differs from tuberculosis in the way that it has no caseous necrosis within the granuloma.

• In most patients, the disease is chronic, involving lymphoid tissue and many other organs including the eye, bones, heart, etc.

• Well-formed granulomas composed of Epitheloid cells and Langhans' giant cells in FNA specimens along with the presence of laminated crystalline inclusions (Schaumann's bodies) in multinucleated giant cells are suggestive of sarcoidosis.

Langhans’ giant cell

Epitheloid cell

Schaumann's bodies

Nocardia in sputum, a loose cluster of long, thin, branching filamentous organisms.

Long filamentous actinomyces

Actinomycosis and Nocardiosis

• Actinomycosis and nocardiosis are suppurative infections caused by gram-positive branching filamentous bacteria .

• Actinomyces grow under conditions of reduced oxygen, and are common inhabitants of the tonsillar crypts and gingival crevices.

• They may be present in the sputum as contaminants of no clinical importance.

• They are readily identified by their growth in colonies made up of dense masses of hematoxylin-stained, tangled filaments that radiate outward and tend to be eosinophilic at the periphery.

• The actinomycotic colonies are visible grossly as small yellow particles (sulfur granules). Nocardia does not form it.

• Nocardia is an aerobic branching filamentous bacterium and is weakly acid fast.

Viral infections

Shallow ulcer in congested mucosa in trachea

Multinucleated cell with basophilic ground glass nuclei

Binucleated bronchial cell with a homogeneous central inclusion within each nucleus, and nuclear clearing about the inclusion with margination of chromatin.

HSV

Fungal infections

Fungi Morphology Diameter Stains D/D

Aspergillus Uniform septate,acute angle branching hyphae

3-6 micron width

PAS,Mucicarmine,Methanamine silver

Candida species

Mucor Broad based,non-septate,right angled branching at irregular interval

6-50 micron in diameter

Grocott-Gomori and methenamine-silver

Other fungi such as Aspergillus

Cryptococcus

Round,thick outer capsule.Narrow based budding

5-10 micron diameter

India ink Blastomyces,histoplasma

Histoplasma

Narrow budding,usually inside the macrophages

Small round 2-5 micron in diameter

Methanamine silver

Crytococcus and Blastomycosis

Candida Small, oval budding yeasts or elongated pseudohyphae forms

2-4 micrometer

PAS Aspergillus

A cluster of cryptococcal spores in sputum

Arrow shows narrow based budding of cryptococcus

Spores & pseudohyphae of candidaAspergillus in sputum showing septate, rather

rigid hyphae branching at an acute angle

Mucor

The hyphae are folded and wavy, flat and broad

compared with aspergillus, and nonseptate. They

branch at right angles compared to the rigid, acute

angle branching of aspergillus.

Squamous cell carcinoma

• Commonest lung cancer in western world.• Is strongly associated with cigarette smoking (>90 %).• M>F• Majority of SCC arise centrally from major bronchi or

segmental bronchi and only 10% occur in periphery.• Originate mainly in the epithelium of secondary or tertiary

bronchi .• Twice as frequent in upper lobes as middle or lower lobes

(upper segment).• Cough, with or without hemoptysis, is by far the most

common clinical symptom.

Differentiating features b/w keratinizing SCC and non-keratinizing SCC

Cytologic features Keratinizing SCC Nonkeratinizing SCC

Cell clusters Less,more discrete cell

More clusters

Cytoplasm Orangeophilic Basophilic

N/C ratio Low High

Nucleoli Absent Prominent

Chromatin Coarse Fine

Pyknotic nuclei Frequent Absent

Fibre and tadpole cells More frequent Less frequent

Squamous cancer cells

Tadpole cell

Ghost cells

SCC (Keratinizing)

Sputum specimen Bronchial brush specimen

Coarse, Irregular hyper chromatic nuclei

Eosinophilic cytoplasmBasophilic cytoplasm

SCC (non keratinizing)

Adenocarcinoma of lung

• Most common lung carcinoma in Asian countries.• It is the most common subtype of lung cancer in females.• Clearly associated with cigarette smoking.• They are commonly located on peripheral part of the lung and

may be detected in an asymptomatic patients.• Four major subtypes of Adenocarcinoma – 1. Acinar2. Papillary Adenocarcinoma of central origin3. Solid 4. Broncholoalveolar • Sputum – more helpful in adenocarcinoma of central origin.• Bronchial brush cytology – more helpful in BA carcinoma.

Clusters of overlapping tumor cells with scanty, pale cytoplasm, relatively large nuclei, finely textured chromatin and prominent nucleoli.

Single cancer cells with abundant finely vacuolated cytoplasm

Sputum & bronchial wash cytology of Adenocarcinoma

The tumor cells have coarsely granular, hyper chromatic nuclei with “nuclear holes” or nuclear cytoplasmic inclusions (arrow). Nucleoli are scarcely visible in these cells.

Glandular formations within the cell cluster S/O Well differentiated Adenocarcinoma.

Bronchial brush cytology of Adenocarcinoma

Sputum showing a cohesive group of small tumor cells with scanty cytoplasm and uniform hyper chromatic nuclei. Sputum with a cluster of glandular cancer

cells that have delicate chromatin, prominent nucleoli and scanty, pale-staining cytoplasm

Sputum cytology of Bronchoalveolar carcinoma type II

Sputum cytology of Bronchoalveolar carcinoma type I

large mucus-secreting, single cancer cells with abundant clear or vacuolated cytoplasm, and large round or ovoid nuclei with delicate chromatin, distinct nuclear membrane and prominent nucleoli

Small cell carcinoma

• In prior classification schemes, these highly aggressive malignant tumors were divided into two subgroups: classical oat cell carcinoma, and an intermediate cell type of SSC.

• Because these two subtypes do not differ clinically, the latest World Health Organization (WHO) classification combines both subtypes as SCC.

• The term combined SSC is used for the not uncommon occurrence of SCC with any non-small-cell component, for example, squamous, adenocarcinoma, or large-cell carcinoma.

A cluster of loosely coherent cells of SSC in a bronchial brush specimen. There is marked variation in cell configuration with molding of adjacent hyper chromatic nuclei.

At low magnification, the loose clusters of small cells can easily be mistaken for lymphocytes.

References

1. Koss' Diagnostic Cytology and Its Histopathology Bases, 5th ed. 2006.

2. Bronchoalveolar Lavage as a Diagnostic Tool SEMINARS IN RESPIRATORY AND CRITICAL CARE MEDICINE VOLUME 28, NUMBER 5 2007

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