This document provides an overview of bronchoalveolar cytology. It discusses the history of exfoliative cytology and various sampling methods used including sputum, bronchial brushings, bronchial washings, and bronchoalveolar lavage. The cytology of normal respiratory tract cells and benign cellular changes are described. Infections that can be identified include pneumonia, tuberculosis, herpes simplex, cytomegalovirus, adenovirus, and various fungal infections. The characteristic cytological findings of these conditions are summarized.

1. BRONCHOALVEOLAR
CYTOLOGY
PRESENTER – DR.NIKITA
MODERATOR – DR.ASHWINI

2. HISTORICAL ASPECT
1845 – Donne, Walshe : presence of tissue fragments of malignant tumor in sputum, exfoliative
cytology.
1860 – Beale : Malignant cells in sputum from a patient with cancer of pharynx
1884-86 – Kronig, Mentrier : used trans-thoracic aspiration
1887 – Hamplen : cancer cells in sputum confirmed to have originated in bronchogenic
carcinoma on autopsy
1970 – Hattori : Fiberoptic bronchoscope

3. SECTIONS
Normal Anatomy
Cytology of the normal respiratory tract
Cytologic sampling methods
Benign cellular changes
Infections
Cytology of malignant lung lesions.

4. ANATOMY
Anatomy and cellular components of the respiratory tract
Upper respiratory tract - 1. nasal cavity 2. paranasal sinuses
3. pharynx
They are lined by stratified squamous and ciliated columnar epithelium.
Lower respiratory tract
1. trachea and bronchi – (a) ciliated columnar cells (b) goblet cells
(c) basal/reserve cells (d) neuroendocrine cells
2. terminal bronchioles
Non ciliated cuboidal/columnar cells (Clara cells)
3. alveoli
(a) type I and II pneumocytes
(b) alveolar macrophages

5. CYTOLOGY OF NORMAL
RESPIRATORY TRACT

6. Cytology of normal
Respiratory Tract
Squamous
Epithelium
Respiratory
Epithelium
Goblet cells
Ciliated cells
Basal or
Germinative
cells
Other epithelial
cells
Fixation
artefacts
Mesothelial
Cells
Alveolar
Macrophages
Leucocytes
Exogenous
Foreign Material
Ferruginous
Bodies
Undigested food
particles
Material of plant
origin
Meat fibres
Pollen
Other
contaminants
Noncellular
endogenous
material
Curshmann’s
spirals
Inspisated
Mucus
Corpora
Amylacea
Amyloid
Calcific
concretions

7. SQUAMOUS EPITHELIUM
Cells are derived from the squamous epithelium of the buccal cavity.
Superficial Squamous cells

8. RESPIRATORY EPITHELIUM
Does not desquamate freely as opposed to the squamous epithelium.
Commonly seen in: specimens from bronchial brushing, aspiration, bronchoscopy.
Uncommon in: Sputum
If present in sputum: indication of prior instrumentation, trauma, severe cough
OR
normal cells from nasal cavity or nasopharynx.

9. RESPIRATORY EPITHELIUM
CILIATED COLUMNAR CELLS:
Singly or in groups or clusters.
Larger bronchi: cells are 30-50 micron in length and
10-15 micron in width.
Terminal bronchi cells: square shaped
Cytoplasm: homogenous, lightly basophilic, granules
of brown lipochrome pigment.
Nuclei: oval in shape with fine chromatin; sex
chromatin may be recognised in females.
Terminal plate for anchoring cilia.
Honeycomb appearance.

10. RESPIRATORY EPITHELIUM
Honeycomb appearance of Bronchial cells Lipochrome pigment

11. RESPIRATORY EPITHELIUM
GOBLET CELLS:
Mucus producing basophilic vacuoles
Less common than ciliated cells.
Basal nucleus and distended supranuclear cytoplasm.
Present in increased number in:
1) Asthma 2) Chronic irritation
BASAL OR GERMINATIVE CELLS:
Source of epithelial regeneration.
Single layer of cells on basement membrane.
Goblet cells

12. ALVEOLAR MACROPHAGES
Spherical or oval cells, 10-25 micron in diameter.
Amphophilic cytoplasm.
Contain phagocytized gray, brown, or black granular dust
particles, also called Dust cells.
Nuclei are generally round, oval, or kidney shaped, 5 to 10
micron in diameter, fine chromatin and small nucleoli.
Binucleation common.

13. LEUKOCYTES
Polymorphonuclear leukocytes:
- Normally present in small numbers.
-If present in large numbers, in presence of necrotic
material in an acutely ill patient, suggests Pneumonia
or abscess.
Eosinophils:
-Allergic process, bronchial asthma
Plasma cells:
-seen in chronic inflammatory processes
Monocytes:
-precursor of large alveolar macrophages
Neutrophils
Eosinophils

14. LEUKOCYTES
Lymphocytes:
-Singly or in pools.
- seen in inflammatory disorders
-Cigarette smokers have increased number of CD8+ T
lymphocytes in the presence of COPD.
Lymphocytes

15. Cytologic Sampling Methods
Sputum
Bronchial
Brushings
Bronchial
Aspirates and
Washings
Bronchoalveolar
Lavage (BAL)
Fine Needle
Aspiration

16. SPUTUM
Sample used: Sputum produced spontaneously (preferably
early morning) or induced.
Coughing can be induced by inhalation of a heated aerosol of
20% polypropylene glycol in hypertonic (10%) saline.
Collected in : a wide mouth container with or without a
fixative

17. SPUTUM
Processing Techniques for fresh and samples prefixed with 50% or 70 % ethanol:
1) PICK & SMEAR : inspect for suspicious material, tissue particles or blood stained areas; smeared between two
microscopic slides and stained.
2) Saccomanno’s technique (mechanical homogenisation).
3) Chemical homogenisation (DTT used)
At KMIO laboratory the PICK & SMEAR technique is used for processing the sputum.
Adequacy:
Sputum specimen should be considered adequate for evaluation if :
Microscopic examination reveals numerous alveolar macrophages.

18. BRONCHIAL BRUSHING AND WASHINGS
Instrument used : Fiberoptic bronchoscope
Sample : obtained with a small brush threaded through a
separate channel in bronchoscope.
Permits sampling of a visualised mucosal abnormality or
systemic sampling of all segmental bronchi to confirm and
localise occult in situ or early invasive carcinomas.
Bronchial washings : better sample, because they can provide
information on the status of the small bronchi beyond the
reach of the bronchoscopic brush.

19. BRONCHIAL BRUSHING AND WASHINGS
Adequacy:
A bronchial washing/brushing specimen is considered satisfactory when:
1) cells or agents diagnostic of a pathologic process are present,
2) a large number of well-preserved, optimally stained ciliated bronchial epithelial cells and
3) Macrophages, goblet cells and a few squamous cells are present
Advantage:
Easier to evaluate the slide, as lesser number of cells, as compared to sputum.
Disadvantage:
1. Procedure is unpleasant for the patient.
2. It has to be performed by a highly skilled physician.

20. BRONCHOALVEOLAR LAVAGE (BAL)
Under local anaesthesia
Bronchoscope passed to the segment of interest
Wedged to occlude the bronchial lumen
100-300ml of NS is instilled in 20-50ml aliquots
Reaspirated
Processed

21. BRONCHOALVEOLAR LAVAGE (BAL)
NON SMOKER
Alveolar macrophages > 85%
Lymphocytes 10-15%
Neutrophils > 1%
Eosinophils > 1%
Ciliated columnar cells > 2%
T4:T8 ratio 0.9-2.5
SMOKER
4 times greater cell yield with slightly
increased number of neutrophils

22. BRONCHOALVEOLAR LAVAGE (BAL)
Criteria for rejection include :
1) paucity of alveolar macrophages on the prepared glass slides
2) excessive numbers of epithelial cells,
3) a mucopurulent exudate of polymorphonuclear cells;
4) numerous red blood cells in combination with at least one of the other criteria for
inadequacy; or
5) degenerative changes or artifacts obscuring cell identity.
In addition, a specimen should be considered adequate if it demonstrates a specific pathologic
process (viral infection, neoplastic, fungal or bacterial disease).

23. NEEDLE ASPIRATION CYTOLOGY
Transbronchial Percutaneous trans-thoracic USG Guided FNA

24. NEEDLE ASPIRATION CYTOLOGY
Adequacy:
The presence of neoplastic cells in a specimen defines it as adequate.
However, there are no universally accepted morphologic criteria defining a specimen as
adequate in the absence of malignant cells.
Complications:
1) Pneumothorax
2) Hemorrhage
3) Air embolism

25. BENIGN CELLULAR
CHANGES

26. BENIGN ABNORMALITIES OF
RESPIRATORY EPITHELIUM
LOSS OF CILIA AND TERMINAL PLATE, due to thermal injury,
viral infection.
MULTINUCLEATION (single cells), non specific reaction to injury.
SYNCTIAL MULTINUCLEATION, possibility of viral
infection.

27. BENIGN ABNORMALITIES OF
RESPIRATORY EPITHELIUM
CYTOMEGALY, KARYOMEGALY & NUCLEOLAR PROMINENCE,
Seen in bronchitis, bacterial or viral pneumonia and in tuberculosis.
CILIOCYTOPHTHORIA,
In viral infections.

28. BENIGN ABNORMALITIES OF
RESPIRATORY EPITHELIUM
BASAL CELL HYPERPLASIA: small, with scanty cytoplasm and relatively large hyperchromatic nuclei.
Seen in Bronchiectases, bacterial and mycotic infections.

29. CREOLA BODIES
Hyperplastic bronchial mucosa sheds spherical or ovoid papillary clusters of bronchial cells.
Presence of cilia or terminal plate, normal goblet cells on the free surface of the cluster.
Bronchial asthma, viral pneumonitis, bronchiectases.

30. SQUAMOUS METAPLASIA
Loosely coherent flat plaque of cuboidal, relatively small cells with amphophilic or eosinophilic cytoplasm.
Adhere well to each other and may have nuclei that are vesicular and open.
Seen in chronic bronchitis, bronchiectasis, lung abscess and granulomatous inflammation and post radio/chemotherapy.

31. ABNORMALITIES OF SQUAMOUS
EPITHELIUM
Inflammatory changes:
•Acute inflammatory process causes necrosis of squamous cells.
•Nuclear pyknosis, karyorhexis and frayed cytoplasm.
•Ulceration/erosison; cells appear in clusters.
•Coarse chromatin and an irregular nuclear membrane.
•Can be caused as a consequence to radiotherapy.

32. ABNORMALITIES OF PULMONARY
MACROPHAGES
Lipid pneumonia: large, multinucleated
macrophage with phagocytized lipid.
‘Heart failure’ cells: hemosiderin in
cytoplasm of macrophages.
Anthracotic pigment : in
pneumoconiosis

33. NON CELLULAR ENDOGENOUS
MATERIAL
CURSCHMANN SPIRAL:
Diagnostic of chronic bronchitis or asthma
MUCUS BLOBS:
Mistaken for nuclei of cancer cells

34. NONCELLULAR ENDOGENOUS MATERIAL
CORPORA AMYLACEA:
Spherical, structureless condensates of protein,
associated with Pulmonary edema.
AMYLOID:
Pale eosinophilic acellular material

35. NONCELLULAR ENDOGENOUS MATERIAL
CALCIFIC CONCRETIONS (PNEUMOLITHS):
Seen in sputum of people with Tuberculosis.

36. EXOGENOUS FOREIGN MATERIAL
FERRUGINOUS BODIES (Asbestos Bodies):
Golden-brown, segmented or beaded bamboo shaped with knobbed
or bulbous ends.
Demonstrated more easily in BAL

37. EXOGENOUS FOREIGN MATERIAL
VEGETABLE (PLANT) CELLS:
Identified by the presence of cellulose walls
which do not stain by Pap or H&E.
POLLEN

38. INFECTIONS

39. PNEUMONIA
Bacterial pneumonia: Atypical pneumonia:
Causes: Causes:
1. Pneumococci 1. Mycoplasma
2. Streptococci 2. Viruses – adeno, rhino, influenza
3. Staphylococci 3. Pneumocystis carinii
4. Klebsiella
5. Legionella

40. PNEUMONIA
Acute and subacute stages:
1. Grossly purulent or blood stained sputum
2. Desquamated ciliated bronchial cells
3. Non specific atypias
4. Predominance of neutrophils
5. Causative organisms
6. Papillary clusters of hyperplastic bronchial
cells i.e. Creola bodies and ciliocytophoria.
Purulent exudate : necrotic cellular material and
Intact and damaged PMN leucocytes.

41. PNEUMONIA
Bronchial cell atypia in viral pneumonia – CREOLA BODIES

42. TUBERCULOSIS
EPITHELIOID CELLS:
Slender, elongated, with carrot shaped nuclei.
MULTINUCLEATED LANGHANS’ GIANT CELL

43. TUBERCULOSIS
FNA SMEAR (HP):
Granulomatous inflammation
ZN Staining : M.tuberculosis

44. HERPES SIMPLEX
Multinucleated cell with nuclear molding
and ground glass nuclei.
Binucleated bronchial cell with single well formed
homogenous eosinophilic nuclear inclusion in each nucleus.
(Cowdry Type A)

45. CYTOMEGALOVIRUS
Large nucleus with basophilic inclusions
surrounded by clear halos
(OWL’S EYE INCLUSIONS)

46. ADENOVIRUS
- Adenovirus is characterised by cytomegaly and
occasional multinucleation.
- Single or multiple basophilic nuclear inclusions with
halos are present.
- The cells retains it columnar shape and cilia and a
terminal bar can be identified.
- No inclusions are seen in the cytoplasm.

47. PULMONARY MYCOSES
Sputum or BAL specimens are commonly used for diagnosis.
Most organisms can be identified in the routinely Pap stained cytologic material from the
respiratory tract.
Pathogenic fungi: Opportunistic fungi:
1) Cryptococcus neoformans 1) Candida albicans
2) Blastomyces dermatitidis 2) Aspergillus Species
3) Coccidioides immitis 3) Mucor Species
4) Paracoccidioides brasiliensis 4) Pneumocystis carinii
5) Histoplasma capsulatum
6) Sporothrix schenkii

48. Cryptococcus neoformans
- observed in AIDS and occasionally in immunosuppresed patients
- lungs are the site of entry for the fungus
CLUSTER OF CRYPTOCOCCAL SPORES:
5-25 micron in diameter, sharply demarcated faint capsule
Tear drop shaped spore attached to the
mother cell by a narrow pedicle

49. Pneumocystis carinii
- ubiquitous organism, associated with AIDS.
- P. carinii pneumonia is often the first and
dominant complication of AIDS.
- On cytology, not very easily identified.
- Presence is signalled by the finding of finely
vacuolated or foamy proteinaceous alveolar casts.
- Vacuoles are due to the presence of unstained
Pneumocystis cysts.
- Spherical, oval, cup-shaped structures with one
flat surface, measuring 4-6 micron in diameter.
- walls stained by GMS or Gram Weigert stain.
PROTEINACEOUS CYST
GRAM WEIGERT STAIN FOR CYSTS

50. Aspergillus species (Aspergillosis)
Rigid, thick, brown septate hyphae
branching at 45 degrees
Conidiospore
Aspergillus niger, forms calcium oxalate crystals, seen in cytologic smears. Presence of crystals alone,
even if organism cannot be identified, is highly suggestive of Aspergillosis.

51. Histoplasma capsulatum
Pulmonary forms of infection can
mimic tuberculosis
Tiny organisms (2-4 micron), within
cytoplasm of macrophage, appear as
tiny dot-like structures with clear
halos.
GMS stain

52. TREATMENT EFFECTS

53. RADIATION THERAPY
Acute radiation changes, observed in squamous cells, not be mistaken
for residual squamous carcinoma.
cellular enlargement with nuclear enlargement;
Enlarged nuclei, have ‘empty’ look with very finely granular chromatin.
Multinucleation, prominent nucleoli, nuclear or cytoplasmic
vacuolations.
Chronic radiation effects are seen months after therapy. Cells show,
slight irregularity, mild hyperchromaisa of nuclei, and cytoplasmic
eosinophilia.
Cellular enlargement, multinucleation,
nuclear vacuolation, prominent nucleoli

54. RADIATION THERAPY
On respiratory epithelium, acute effects are seen as in
multinuceation of ciliated bronchial cells.
marked enlargement of all cellular components with
preservation of N:C ratio.
Intranuclear cytoplasmic inclusions are also seen.
The finding of bizzare giant cells is more commonly caused by
radiation than by the uncommon giant cell carcinoma.
Chronic radiation effects include enlargement of Type II
pneumocytes and squamous metaplasia
Cellular enlargement, multinucleation and
loss of chromatin structure

55. CHEMOTHERAPY
very large cells with large
hyperchromatic or vesicular nuclei.
atypia of bronchial epithelium.
Induces keratinization and death of squamous cells and
form MNG’s that phagocytize keratin.

56. CYTOLOGY OF
MALIGNANT LESIONS
OF LUNG

57. WHO CLASSIFICATION OF TUMORS OF THE LUNG
•Adenocarcinoma
•Squamous cell carcinoma – 1. Keratinizing 2. Non-keratinizing 3. Basaloid 4. Preinvasive
•Neuroendocrine – 1. Small cell carcinoma 2. Large cell neuroendocrine carcinoma 3.
Carcinoid tumors 4. Preinvasive lesion
•Large cell carcinoma
Epithelial tumors
•Pulmonary hamartoma
•Chondroma
•PEComatous tumors
Mesenchymal tumors
•MALT lymphoma
•DLBCL
•Lymphomatoid granulomatosis
Lymphohistiocytic
tumors
•Germ cell tumors – Teratoma (mature and immature)
•Intrapulmonary thymoma
•Melanoma
Tumors of ectopic origin
Metastatic tumors

58. ADENOCARCINOMA
Associated with cigarette smoking.
Could be central or peripheral.
Large, round or polygonal cells, occasionally columnar, arranged
in glandular or acinar pattern.
Moderate cytoplasm, finely vacuolated and faint staining,
usually basophilic.
Large nuceli, finely granular chromatin, slight to moderate
hyperchromasia, with prominent single or multiple nucleoli

59. ADENOCARCINOMA

60. SQUAMOUS CARCINOMA
Considerable variations in size and shape with a
background of inflammation and necrosis.
Cells vary in shape and size, have deeply eosinophilic cytoplasm,
and relatively large, hyperchromatic nuclei without discernable internal structure (‘ink spot’ nuclei).

61. SQUAMOUS CARCINOMA
Tadpole cells

62. NEUROENDOCRINE TUMORS
Small cell carcinoma
Large cell neuroendocrine carcinoma
Carcinoid tumors
Preinvasive lesion

63. SMALL CELL CARCINOMA
Small cancer cells can be misinterpreted as lymphocytes.
Loosely arranged clusters of small cells of variable sizes.
Relatively large nuclei, scanty basophilic rim of cytoplasm.
Molding of adjacent nuclei in clusters is seen.
Frequent mitoses.

64. SMALL CELL CARCINOMA
Pyknotic nuclei – necrotic parts
Vesicular nuclei – non necrotic
Fragile nucleus
Crushing artefact – smudges and streaks of
nuclear material is of diagnostic value since it is
uncommon in other tumors; AZZOPARDI EFFECT

65. LARGE CELL NEUROENDOCRINE
CARCINOMA
Round or ovoid nucleus with irregular nuclear membrane.
Finely divided hyperchromatic nuclear chromatin.
More abundant cytoplasm as compared to small cell lung
carcinoma.
Visible nucleolus
Distinguished from other tumors by expression of neuroendocrine
markers in immunocytochemical stains for chromogranin and
synaptophysin.

66. CARCINOID TUMORS
Discohesive with fragments of capillaries in background.
Necrosis is absent and the background is clean
Small cells with round, oval or spindle shaped nuclei.
Finely granular chromatin with inconspicuous nucleoli.
Rosette shapes may be seen.

67. LARGE CELL CARCINOMA
Undifferentiated non-small cell carcinoma that lacks the
cytological, architectural and immunohistochemical
features of squamous, adeno and small cell carcinoma.
Syncytial clusters and dispersed cells with
• irregular nuclei
• striking chromatin clearing
• prominent, often multiple nucleoli
• ill-defined, feathery cytoplasm

68. MOLECLAR PATHOLOGY OF NON SMALL
CELL LUNG CARCINOMA
Distinction between small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC) is no
longer sufficient for treatment planning.
 Molecular characterization of lung carcinoma contributes valuable information in terms of the
diagnosis, prognosis, and the potential for treatment with targeted therapy.
In cytology, the fine needle aspirates, cell blocks and liquids are suitable for molecular testing.
In KMIO, of the 129 cases of IHC on cell block in the year 2013, the lung and pleura cases
comprised of 15.
The cell block was used for IHC studies and then also available for further molecular testing.

69. MOLECLAR PATHOLOGY OF NON SMALL
CELL LUNG CARCINOMA
EGFR, KRAS & ALK are considered the mutations in lung carcinoma.
Most common in adenocarcinoma
EGFR & ALK, seen in non smokers and develops in peripheral lung parenchyma.
KRAS mutations are seen in smokers and in the hilar region and are seen in squamous
carcinoma and small cell carcinoma.

70. SUMMARY
Anatomy
Cytology of normal respiratory tract
-Squamous epithelium
-Respiratory epithelium – (a) ciliated columnar cells (b) goblet cells (c) basal cells
-Alveolar macrophages
-Leucocytes
-Non-cellular endogenous material
-Exogenous foreign material

71. SUMMARY
Cytologic sampling methods
-Sputum
-Bronchial brushing
-Bronchial aspiration and washing
-Bronchoalveolar lavage
-Needle aspiration cytology

72. SUMMARY
Benign cellular changes:
1. Abnormalities of the respiratory epithelium
(a) Basal cell hyperplasia
(b) Papillary hyperplasia (Creola bodies)
(c) Loss of cilia and terminal plate, multinucleation, cell and nuclear enlargement,
ciliocytophhthoria.
(d) Squamous metaplasia
2. Abnormalities of the squamous epithelium
(a) Inflammatory changes

73. SUMMARY
3. Abnormalities of alveolar macrophages
(a) Nuclear abnormalities
(b) Lipid pneumonia
(c) Heart Failure cells
Infections
1. Bacterial : pneumonia, Tuberculosis
2. Viral
3. Pulmonary Mycoses

74. SUMMARY
 Treatment effects
1. Radiation therapy
2. Chemotherapy
Cytology of malignant lesions of the lungs
1. Adenocarcinoma
2. Squamous carcinoma
3. Neuroendocrine tumors
4. Large cell carcinoma

75. REFERENCES
KOSS’ DIAGNOSTIC CYTOLOGY, 5TH edition : Chapter 19,20 : 568-712
CYTOLOGY DIAGNOSTIC PRINCIPLES AND CLINICAL CORRELATES, 4th edition : Chapter 2 : 59-104
WHO CLASSIFICATION OF TUMORS OF THE LUNG, PLEURA, THYMUS AND HEART, 4TH edition
DAIL AND HAMMAR’S PULMONARY PATHOLOGY : Chapter 29 :1030-1031
K. Khetani, M. Weir. CSC Guidelines for reporting results on cytological specimen from the
respiratory tract, 2011
M. Heron, J.Grutters et al. Bronchoalveolar lavage cell pattern from healthy human lung. Clinical
and Experimental Immunology, 2011; 167 : 523-531
Sharma R, Desai H, Malukani P et al. Comparison of bronchoalveolar lavage cytology and biopsy
in lung malignancy. Int J Cur Res Rev, 2014; 06(05) : 43-47

76. REFERENCES
Hubers A, Prinsen C, Sozzi G, et al. Molecular Sputum Analysis for the diagnosis of lung cancer.
Bristish Journal of Cancer, 2013 ; 109 : 530-537
Rao S, Lal A, Barathi G, et al. Bronchial wash cytology: A study on morphology and
morphometry. J Cytol, 2014 ; 31(02) : 63-67
Zimpfer A, Polak A, Bier A, et al. Assessment of Diagnostic Accuracy of Bronchoalveolar Lavage
Cytology in the Diagnosis of Lung Tumors and Contribution to the Classification of Non-Small Cell
Lung Cancer Entities: A Retrospective Clinocopathological Study. OJPathology, 2013 ;3 : 107-112

77. THANK YOU