This document provides an overview of cytologic assessment of bronchopulmonary lesions. It discusses the normal histology of the respiratory system and various cytologic sampling techniques used to evaluate the lungs, such as sputum samples, bronchial brushings, washings and lavage. The cytology of normal respiratory cells and endogenous material is described. A variety of benign pulmonary conditions and infectious processes are outlined. Specific lung diseases like tuberculosis, sarcoidosis and nocardiosis are discussed through their characteristic cytologic findings.

1. Cytologic assessment of
bronchopulmonary lesions
Malvika tripathi
Resident of pathology

2. 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.

3. Embryology

4. Anatomy

5. 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.

7. • 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.

8. • 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.

9. 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.

10. 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.

11. • 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.

12. Processing of sputum
• Pick & Smear technique -

13. 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.

14. 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.

17. • 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.

18. 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.

19. 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.

20. • Uses of BAL –

21. 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.

22. • 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.

26. Processing and lab assessment of BAL
Process 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 fluid
2. Absolute number of total nucleated cells and total number of red blood cells
3. White blood cell differential percentages
4. Percentage of epithelial cells that represent total nucleated cells
5. Other specific findings (e.g., plasma cells, mast cells, foamy alveolar
macrophages.

28. 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.

29. • 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

30. • 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.

31. • 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

32. 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.

34. • 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.

35. • 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.

36. • 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.

37. 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.

38. • 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.

39. • 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.

40. Alveolar macrophages

41. • 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.

42. • 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.

43. 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.

45. • Non cellular endogenous material
Curschmann's Spirals
Mucus blobs / Inspissated
mucus
Corpora amylacea

46. 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

47. Other benign conditions
1. Alveolar proteinosis
2. Malakoplakia
3. Rheumatoid granuloma
4. Gaucher disease
5. Sclerosing hemangioma
6. Follicular bronchitis
Tumors of lung
1. Squamous cell carcinoma
- Well differentiated /Keratinizing
- Poorly differentiated/ Non keratinizing
2. Large cell undifferentiated carcinoma
3. Small cell undifferentiated carcinoma
- Oat cell
- Intermediate cell type
4. Adenocarcinoma
- Adenocarcinoma of central origin
- Bronchoalveolar carcinoma
5. Adenosquamous carcinoma
6. Mucoepidermoid carcinoma
7. Spindle and giant cell carcinoma
8. Neuroendocrine tumors

48. 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.

49. Langhans’ giant cell
Epitheloid cell

50. Cluster of Epitheloid cells and spindle cells suggestive
of granuloma

51. 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.

52. Langhans’ giant cell
Epitheloid cell
Schaumann's bodies

53. Nocardia in sputum, a loose cluster of
long, thin, branching filamentous
organisms.
Long filamentous
actinomyces
Actinomycosis and Nocardiosis

54. • 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.

55. Viral
infections

56. 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

57. Fungal infections

58. Fungi Morphology Diameter Stains D/D
Aspergillus Uniform
septate,acute angle
branching hyphae
3-6 micron
width
PAS,Mucicarmi
ne,Methanami
ne 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
Cryptococc
us
Round,thick outer
capsule.Narrow
based budding
5-10 micron
diameter
India ink Blastomyces,his
toplasma
Histoplasm
a
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

59. A cluster of
cryptococcal
spores in
sputum
Arrow shows
narrow based
budding of
cryptococcus

60. Spores & pseudohyphae of candida
Aspergillus 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.

61. 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.

62. 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

63. Squamous cancer cells
Tadpole
cell
Ghost
cells
SCC (Keratinizing)

64. Sputum specimen Bronchial brush specimen
Coarse, Irregular hyper chromatic nuclei
Eosinophilic cytoplasm
Basophilic cytoplasm
SCC (non keratinizing)

65. 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. Acinar
2. Papillary Adenocarcinoma of central origin
3. Solid
4. Broncholoalveolar
• Sputum – more helpful in adenocarcinoma of central origin.
• Bronchial brush cytology – more helpful in BA carcinoma.

66. 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

67. 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

68. 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

69. 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

70. 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.

71. 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.

72. 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

73. Thank you