BRONCHOALVEOLAR CYTOLOGY.pptx

pathology

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

7. 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.

8. 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.

9. RESPIRATORY EPITHELIUM Honeycomb appearance of Bronchial cells Lipochrome pigment

10. 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

11. 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 a re generally round, oval, or kidney shaped, 5 to 10 micron in diameter, fine chromatin and small nucleoli. B inucleation common.

12. 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

13. 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

14. 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

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

16. 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.

17. BRONCHIAL BRUSHING AND WASHINGS Adequacy: A bronchial washing/brushing specimen is considered satisfactory when: cells or agents diagnostic of a pathologic process are present , a large number of well-preserved, optimally stained ciliated bronchial epithelial cells and 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: Procedure is unpleasant for the patient. It has to be performed by a highly skilled physician.

18. 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 P rocessed

19. 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

20. BRONCHOALVEOLAR LAVAGE (BAL) Criteria for rejection include : paucity of alveolar macrophages on the prepared glass slides excessive numbers of epithelial cells , a mucopurulent exudate of polymorphonuclear cells ; numerous red blood cells in combination with at least one of the other criteria for inadequacy; or 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).

21. NEEDLE ASPIRATION CYTOLOGY T ransbronchial Percutaneous trans-thoracic USG Guided FNA

22. 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: Pneumothorax Hemorrhage Air embolism

23. BENIGN CELLULAR CHANGES

24. 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.

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

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

27. 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 .

28. 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.

29. 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.

30. ABNORMALITIES OF PULMONARY MACROPHAGES Lipid pneumonia: large, multinucleated m acrophage with phagocytized lipid. ‘Heart failure’ cells: hemosiderin in c ytoplasm of macrophages. Anthracotic pigment : in pneumoconiosis

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

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

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

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

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

36. INFECTIONS

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

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

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

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

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

42. HERPES SIMPLEX Multinucleated cell with nuclear molding a nd ground glass nuclei. Binucleated bronchial cell with single well formed h omogenous eosinophilic nuclear inclusion in each nucleus. ( Cowdry Type A)

43. CYTOMEGALOVIRUS Large nucleus with basophilic inclusions s urrounded by clear halos (OWL’S EYE INCLUSIONS)

44. 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.

45. 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: Cryptococcus neoformans 1) Candida albicans Blastomyces dermatitidis 2) Aspergillus Species Coccidioides immitis 3) Mucor Species Paracoccidioides brasiliensis 4) Pneumocystis carinii Histoplasma capsulatum Sporothrix schenkii

46. 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

47. Pneumocystis carinii - ubiquitous organism, associated with AIDS. - P. carinii pneumonia is often the first and dominant complication of AIDS. - O n 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

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

49. 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

50. TREATMENT EFFECTS

51. 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 , n uclear vacuolation , prominent nucleoli

52. 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

53. CHEMOTHERAPY very large cells with large h yperchromatic or vesicular nuclei . atypia of bronchial epithelium. Induces keratinization and death of squamous cells and f orm MNG’s that phagocytize keratin.

54. CYTOLOGY OF MALIGNANT LESIONS OF LUNG

55. WHO CLASSIFICATION OF TUMORS OF THE LUNG

56. 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

57. ADENOCARCINOMA

58. 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, a nd relatively large, hyperchromatic nuclei without discernable internal structure (‘ink spot’ nuclei).

59. SQUAMOUS CARCINOMA Tadpole cells

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

61. 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.

62. 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

63. 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 .

64. 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.

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

66. 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 .

67. 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.

68. 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

69. SUMMARY Cytologic sampling methods Sputum Bronchial brushing Bronchial aspiration and washing Bronchoalveolar lavage Needle aspiration cytology

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

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

72. SUMMARY Treatment effects Radiation therapy Chemotherapy Cytology of malignant lesions of the lungs Adenocarcinoma Squamous carcinoma Neuroendocrine tumors Large cell carcinoma

73. REFERENCES KOSS’ DIAGNOSTIC CYTOLOGY, 5 TH edition : Chapter 19,20 : 568-712 CYTOLOGY DIAGNOSTIC PRINCIPLES AND CLINICAL CORRELATES, 4 th edition : Chapter 2 : 59-104 WHO CLASSIFICATION OF TUMORS OF THE LUNG, PLEURA, THYMUS AND HEART, 4 TH 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

74. 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

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