L8 - Respiratory Cytology Flashcards
Cytology
- The study and identification of disease by light microscopy examination of celluar material
- Used as a diagnostic tool as well as a screening test
- The main role of diagnostic cytology in pathology is the identification of malignant or premalignant cells by recognising changes in cell morpholgy
Types of samples
- Exfoliative
- Fine needle aspiration
- Abrasive
Histology of respiratory tract
Respiratory epithelium: pseudostratified columnar
- Ciliated
- Muscus secreting goblet cells
- Basal or reserve cells
- Non ciliated and cuboidal cells in samller bronchi
- Clara cells: produce surfactant (specialised cells that line the bronchioles)
- Kulchitsky cells: are specialised neuroendocrine cells
- Alveoli
- Macrophages
Alveoli
- gas exchanging structure of the lungs
Lined by two types of epitelium:
- Type 1 pneumocytes: thin walled and have a large surface area, a combination that facilitates gas exchange
- Type 2 pneumocytes: are more cuboidal and secrete surfactant
Pulmonary macrophages
large phagocytic cells containing dust particles
Sputum samples
- traditional specimen
- early morning deep cough
- if a specimen cannot be produced spontaneously, representative samples may be obtained during physiotherapy or through nebulizer induced deep cough
Advantages of sputum samples
- Easily obtained
- Extensive area sampled
- Good for central tumours
- accurate diagnosis
- best screening test
Disadvantages of sputum samples
- difficult to obtain if not spontaneous
- cannot localise lesion
- poor for peripheral lesions
- benign tumours difficult to diagnose
- less accurate for adeno carcinoma, metastasis and lymphomas
Bronchial washings
- obtained useing a flexible fibre-optic bronchoscope
- samples are mucoid in consistency and material is suspended in saline
- preparation from these samples requires centrifugation at 1500rpm for 10 min to sediment deposit
- indications for bronchoscopy indclude cough, haemoptysis, bronchial obstruction, and partial lung collapse
Bronchial brushings
- material is directly brushed or scraped from a lesion during a bronchoscopy
- brushings have a higher diagnostic yield for metastatic carcinoma, peripheral tumours and large necrotic cancers
- usually clinical suspicion of infection warranting special staining procedures (e.g. methanamine silver for fungi)
- overall sensitivity of bronchial brushing is 70%
Bronchiolar-alveolar lavage (BAL)
- performed for the assessment and monitoring of interstitial lung disease
- a useful tool for obtaining material for cytology and microbiology from immunocompromised individuals
- performed when saline is instilled and aspirated in serial washes through a fibre-optic
Fine needle aspiration (FNA)
- material is directly removed through aspiration of a subcutaneous lesion using a syringe
- percutaneous (most common) and transbronchial
- skin, chest wall and pleural anaesthetised
- under CT/US guided imaging
- slides are stained (ROSE - rapid onsite evaluation) by H&E stain and examined immediately to determine specimen adequacy
Possible complications for FNA
- pneumothorax
- haemorrage
- air embolism
- needle track seeding
Epithelial cells in sputum and respiratory samples
- non keratinised stratified squamous epithelial cells
Respiratory bronchial epithelial cells:
- ciliated columnar cells
- columnar cells in sheets and groups
- goblet cells
- multinucleated bronchial cells
Bronchial epithelium cells
- less common in sputum samples
- readily seen in post bronchoscopy samples
- shed in sheets and cohesive aggregates
- tall columnar appearance with a basophilic homogenous cytoplasm
- terminal plate may be visible on the apical surface with or without cilia
Bronchial epithelium cells - pneumocytes
- type 1 alveolar pneumocytes are flat cells which cover 90% of the alveolar surface
- these are not usually identified on cytology samples
- type 2 alveolar pneumocytes are columnar cells that are normally found scattered in the alveoli and secrete surfactant
- usually recognised when they become hyperplastic
Cells of non-epithelial origin
- alveolar macrophages
- pulmonary macrophages
- lymphocytes
- eosinophils
Cells of non-epithelial origin: Alveolar macrophages
- bone marrow derived histocytes found free in the alveolar space
- hallmark of a satisfactory sputum sample
- similar to histocytes
- vary in size; often have round to oval or bean shaped nucleui, may be mononucleated, bi-nucleated or multi-nucleated
- multinucleated macrophages are often found in granulomatous disease such as sarcoid and TB but are not specific for granulomas
- are characteristically phagocytic, carbon. haemosiderin and lipid
Non - celluar elements
Curshmann spirals
Ferruginous bodies
Alveolar proteinosis
Corpora amylacea
Curshmann spirals
casts of small bronchioles fromed by impacted mucin, found in excess mucus production (e.g. asthma)
Ferruginous bodies
form when iron salts precipitate into tiny rounded or fibrous inhaled dust; the fibre is often asbestos but it can be fibre glass
Alveolar proteinosis
due to an enzymatic disorder of macrophages, results in coarsely granular, PAS+ debris
Corpora amylacea
concentrically laminated calcified bodies, associated with BAC but also seen in TB
Non - specific reactive changes
- reactive sqaumous cells
- anucleated keratinised sqaumous cells
- hyperplasia of bronchial epithelium
- sqaumous metaplasia
Asthma
- during an acute attack there is extensive loss of bronchial epithelium associated with extensive mucous and serous fluid into the bronchial lumen
- eosinophils are present in this exudate which forms mucoid plugs in the airways
Cytological findings of asthma
- visible mucus plugs seen in lavage fluid
- bronchial cells may appear in clusters ‘creola bodies’
- eosinphils and charcot leyden crystals
- cruschmann spirals
- inflamatory debris may contain fungal hyphae
Tuberculosis
- granulomatous reaction: acute or suppurative
- caseous necrotic debris
- mixed inflammatory exudate
- epitheloid histocytes
- langhans giant cells
- AFB organism demonstrated by ZN stain
Fungal and bacterial infections
- actinomyces
- candida
- cryptococcosis
- aspergillus
- mucor
- nocardia
- histoplasma
Aspergillus
- a fungus that causes a variety of clinical syndromes by interacting with the host
- pulmonary aspergillosis is caused by aspergillus fumigatus
- pulmonary aspergillosis is a major cause of infectious mortality in immunocompromised patients
- about 30% of patients with IPA will die from the disease
Clinical syndromes of Aspergillus
- Invasive pulmonary aspergillosis (IPA)
- Chronic pulmoary aspergillosis (CPA)
- Allergic bronchopulmonary aspergillosis (ABPA)
- Aspergilloma
Cytological features of Aspergillosis
- septate fungal organism with hyphae that is 3 - 4 um in width
- dichotomous branching: 45 degree angle
- fruiting heads form in aerobic conditions
- stained by papanicolaou and grocott methods
- branches are refractile under polarized light microscopy
Two main forms of Cryptococcosis
- Cryptococcosis gattii: restricted geopgraphic distribution
- Cryptococcosis neoformans: causes human disease especially in immunocomproised individuals
Cryptococcus neoformans
- an environmental fungus
- opportunistic pathogen, causing focal and disseminated infection
- commonly found in soil , pigeon droppings, eucalyptus trees and decaying wood
- cause infection in lungs and meninges
Diagnostic tools of cryptococcus neoformans
- culture
- histology
- cryptococcal antigen
- diagnostic cytology
Features of cryptococcosis
- an encapsulated yeast that may have budding but no true hyphae
- a single bud may be seen attached to a narrow isthmus
- non-encapsulated forms can occur in AIDS patients
- thick mucoid capsule stains pale and translucent with pap and MGG stains
- stains magenta with
PAS and mucicarmine stains
How Herpes simplex virus happens
- HSV of the respiratory tract can occur anywhere from the oral cavity to the alveoli
- radiographic evaluation and bronchoscopy can be used to detect it
- presence of HSV in the lungs is associated with a poor clinical outcome
Risks of infection for HSV
- immunocompromised patients
- prolonged intubation
- burns
- neonates
- neoplasia
How a diagnosis is made for HSV
Made with:
- cytological material
- sputum
- BrBr
- BrWsh
- BAL
Further clarification:
- IHC
- molecular techniques (ISH)
- cultures
Herpes simplex virus appearance
- multinucleation
- nuclear moulding
- ‘ground glass’ appearance
- loss of chromatin pattern
- nuclear inclusions
- clean background
- exclude contaminants from upper respiratory tract
Pneumocystis Jirovecii
- an opportunistic organism
- results in life-threatening pneumonia in patients who are immunocompromised
- infection occurs but patient is asymptomatic because of the organism’s low virulence
- about 65-100% of children have antibodies against P. jirovecii by the time they are 4 y.o.
Symptoms of P. jirovecii
- fever
- dry cough
- shortness of breath
- dyspnoea on exertion and onset/progression over several weeks
What is seen in P. jirovecii
- commonly seen in immunocompromised patients, premature babies and malnourished infants
- chest xray shows bilateral infiltrates
- alveolar casts
- honeycomb appearance of unstained cysts with pap stain
- casts are 5-8um in diameter
- trophozoites outside cyst are not visible
- p jirovecii can lead to death if not treated
Special stains for pneumocystis
- MGG
- Methanamine silver
- Cresyl violet
- Toludine blue
- Acridine orange stain
- Immunocytochemistry
- EM
Laboratory and diagnosis tests for P. jirovecii pneumocystis
- blood chemistry
- arterial blood gases
- serologies, antigen testing and molecular typing
- sputum (least invasive test)
- BrWash, BAL and transbronchial biopsy (diagnosis yield 82-94%)
- open lung biopsy (increased risk for morbidity)
Pulmonary neoplasms
- multiple sputum samples are best in detecting lesions which arise centrally in the lung such as squamous cell carcinoma and small cell carcinoma
- peripheral and subpleural lesions are best sampled using bronchial brush and wash specimens, bronchoalveolar lavage and fine needle aspiration
Lung cancer
- primary cause of cancer related deaths in developed countries in both males and females
- increasing incidence in females, slowing increasing of incidence in males
- peak incidence: 40 to 70 years
- male to female ratio is around 2:1
Lung cancer in Australia
prognosis for those diagnosed with lung cancer is poor; 5 year relative survival of 11% for males and 15% for females
Most commonly diagnosed cancers for women
- Breast
- Bowel
- Melanoma
- Lung
Most commonly diagnosed cancers for males
- Prostate
- Bowel
- Melanoma
- Lung
Risk factors for lung cancer
- Smoking (largest single cause, responsible for 90% of lung cancer in males and 65% in women)
- smoking increases risk of lung cancer to be 20x greater comparing to those who don’t smoke
- passive smoking: 2x risk in non-smokers
- pipes and cigars: risk is increased but much less, usually associated with upper respiratory tract lesions
Non-smoking risk factors for lung cancer
- radon gas
- exposure to industrial and chemical carcinogens
- air pollution
- family history of lung cancer
- previous lung diseases (e.g. emphysema, lung fibrosis, TB)
How smoking cigarettes causes lung cancer
- progressive alteration in epithelium lining the respiratory tract in habitual smokers
When smoking:
Sqaumous metaplasia»_space; dysplasia»_space; CIS»_space; Invasive carcinoma
Primary lung cancer
- 95% of primary lung cancers are derived from bronchial epithelium (bronchogenic carcinoma)
5% fall into the miscellaneous category and include:
- Bronchial carcinoma tumour
- Mesothelioma
- Mesenchymal tumours
- Lymphomas
Bronchogenic carcinoma
4 major subtypes:
- Non small cell lung carcinoma (NSCLC)
- Squamous cell carcinoma (SCC)
- Adenocarcinoma (ACA)
- Large cell undifferentiated carcinoma (LCLC)
(also small cell carcinoma but not part of the 4 major types)
Cytology of squamous cell carcinoma
- pleomorphism
- lots of single cells
- irregular chromatin
- karyopyknosis
- irregular keratinisation
- irregularly thin cytoplasm and caudate (tadpole) cells
- background of necrosis
SCC differential diagnosis
- repair
- squamous metaplasia
- cavitating lung infections
- pulmonary infarction
- mesothelial cells
- vegetable cells
- radiation/chemotherapy effect
- contamination by carcinoma upper airway
False positive and false negative differential diagnoses for SCC
- False pos diff diagnosis: overinterpretation of reparative or metaplastic changes
- False neg diff diagnosis:
there is only necrotic/inflammatory material, or granulomatous reaction to keratin
Histology of adenocarcinoma
Subtypes:
- acinar
- papillary
- bronchioalveolar subtypes ( a pattern defined as growth of cuboidal cells or columnar tumour cells along alveolar or fibrovascular septa)
- mixed acinar and papillary
- majority of adenocarcinomas develop on the periphery of the lung and may involve pleura
Cytology of lipidic adenocarcinoma
- ball - like clusters of cells
- papillary fronds
- nuclei is round/oval with fine granular chromatin
- nucleoli that is not well seen
- single cells can look a lot like alveolar macrophages, but a lot of these cells will be present
- may have secretory vacuoles
Small cell anaplastic carcinoma
- accounts for 20% of all lung cancers and strongly associated with smoking
- very small cells arranged in loose clusters with some dispersed single cells
- nuclei are round or irregular
- hyperchromatic and dense chromatin
- karyopyknosis
- barely any cytoplasm
- abundant background necrosis
Differential diagnosis for small cell carcinoma
- lymphocytes
- lymphoma
- degenerate bronchial cells
- poorly differentiated squamous cell carc. or non small cell lung carc.
Large cell undifferentiated carcinoma
- mixture of large single cells and a multinucleated mass of cytoplasm that is not separated into cells
- nuclei round to lobulated with irregularly dispersed, very hyperchromatic chromatin
- macronucleoli, may be more than one
- tumour giant cells
- poorly defined cytoplasmic outline
Why is diagnosis so important?
- molecular correlations for lung adenocarcinomas are continually evolving
- the only strong molecular correlation for the predominant subtype of adenocarcinoma is currently a KRAS mutation for invasive mucinous adenocarcinoma, which is EGFR negative
Treatments available for adenocarcinomas with certain molecular features
- tyrosine kinase inhibitors (erlotinib and gefitinib): first treatment options for lung adeno that has EGFR mutations
- adenos with ALK rearrangements are responsive to crizotinib
