Respiratory Tract
Learning Unit 1 – Part 1
Anatomy
• The primary function of respiratory
system is to supply blood with
oxygen and remove carbon dioxide
(gaseous exchange)
• The respiratory system is divided
into upper and lower portions. The
upper respiratory system consists
of the nasal cavities, sinuses,
mouth, pharynx, and larynx.
• The lower respiratory system
consists of the trachea, bronchi,
bronchioles, and alveoli.
Upper respiratory tract
Anatomy
• Air enters the respiratory system through the mouth and the nose.
• The air then passes through the nasal cavity and pharynx, through the
larynx and the trachea.
• The trachea splits into two smaller tubes, left and right main bronchi.
• The lungs differ anatomically in that the left lung has two lobes but
the right lung has three lobes.
• The right bronchus divides into three lobar bronchi, to supply the
upper, middle, and lower lobe of the right lung, and the left bronchus
branches to supply the upper and the lower lobe of the left lung.
Anatomy
Anatomy
• The bronchial tubes divide further into many smaller tubes, which
connect to tiny sacs called alveoli.
• The alveoli are the basic functional component of the lungs and are
surrounded by capillaries.
• Oxygen present in inhaled air passes into the alveoli and diffuses
through the capillaries into the arterial blood. Meanwhile, carbon
dioxide carried in the blood from the veins is released into the alveoli.
• Carbon dioxide follows the same path out of the lungs during
exhalation.
Function of the Respiratory Tract
Upper respiratory tract:
• Transportation of air to lungs
• Warming and humidifying
inspired air
• Removing dust particles
• Voice production
• Plays a role in olfaction
• First line of defence against
inhaled microorganisms
Lower respiratory system:
• Warms, moistens and filters the
air before it reaches the lungs
• Protects against microorganisms
• Terminal portion responsible for
gaseous exchange
Anatomy and Histology
Respiratory system is lined by two types of epithelium:
• Non-keratinizing stratified Squamous Epithelium
• Pseudostratified Columnar (Respiratory) Epithelium
Non-keratinizing Stratified Squamous
Epithelium
• Non-keratinizing squamous epithelium protects & covers areas that
are liable to frictional forces such as those caused by swallowing of
food or by touch.
• These areas include the front part of the nasal cavity, central and
lower portions of pharynx, oral cavity and parts of the larynx.
• Non-keratinizing squamous epithelium cells commonly exfoliate, will
be commonly present in sputum and bronchial specimens.
• They can be recognized in respiratory samples as superficial and
intermediate squamous and parabasal cells.
Pseudo-Stratified Columnar Epithelium
• Respiratory epithelium is referred to as pseudostratified columnar
epithelium as it appears to be multilayered, even though it consists of
a single layer of epithelial cells.
• The impression of multilayering is due to the position of nuclei in
different levels of the cells in the epithelium.
• The trachea, bronchial tree and remainder of the upper airways are
lined by specialize columnar epithelium.
• This consists of a pseudostratified layer of ciliated tall columnar cells
interspersed with mucin secreting goblet cells, which have microvilli
on their luminal surfaces.
Pseudo-Stratified Respiratory Epithelium
• Bronchioles are lined by a single layer of columnar or cuboidal cells
epithelium composed of three cell types:
➢ Ciliated columnar cells
➢Mucous – secreting goblet cells
➢Basal or reserve cells (precursors for goblet and columnar cells)
➢ Non- ciliated non mucous secreting Clara cells and Neuro-endocrine cells
(line the terminal bronchioles).
Cytology of Normal Respiratory Tract
Squamous Epithelial Cells
• Squamous mucosa is composed of
basal, parabasal, intermediate &
superficial cells
• Superficial and intermediate cells
predominate have well-defined cell
borders, abundant cytoplasm
staining pinkish orange and green
respectively, in Pap stained smears.
• Superficial cells characterised by
pyknotic nuclei.
• Intermediate cells are characterized
by a round-to-oval vesicular nuclei.
Ciliated Columnar cells
• These cells seen in bronchial brushings/washings, BALs and FNABs.
• Not present in large numbers in sputum except in post- bronchoscopy
specimens or following extensive damage to the respiratory
epithelium.
• The main function of ciliated cells is to move the bronchial secretions
toward the pharynx.
Ciliated Columnar Cells
• Cells lie singly, in short ribbons or
flat sheets which have a
honeycomb appearance.
• The ciliated cells have basal, round
to oval shaped nuclei with finely
granular (open) chromatin.
• The luminal part (facing the lumen)
is covered by cilia; these are
numerous and at their point of
attachment form a terminal plate
• Cells have delicate/foamy
cyanophilic cytoplasm
Ciliated bronchial epithelial cells
Goblet Cells
• Goblet cells are present in a ratio of approximately one per six ciliated
cells.
• Cells less commonly seen in sputum specimens.
• Goblet cells secretes mucous which coats the airways with a sticky
layer within which inhaled particles, organisms and cell debris are
trapped
• Common in patient with chronic tracheobronchial disease, such as
asthmatic bronchitis, chronic bronchitis and bronchiesctasis
(permanent dilation of one or more bronchi)
Mucous Secreting Goblet Cells
• Cells are columnar in shape.
• Also have a basally located
nucleus, fine vesicular chromatin
but they lack cilia.
• Cells have microvilli on luminal
surface.
• Cytoplasm is distended by
single/multiple vacuoles filled
with mucus.
Goblet Cells
Reserve Cells
• Small reserve cells rest on the basement
membrane, forming an undifferentiated
stem cell population from which
regeneration of goblet & columnar cells
takes place after injury.
• They are rarely present in sputum but are
sometimes seen in brushings and lavage
specimens, mainly in reactive states.
• They form sheets of small regular cells
slightly larger than lymphocytes, with a
high nuclear/cytoplasmic ratio, coarse
chromatin and a narrow rim of green
cytoplasm.
Reserve Cells
Neuroendocrine Cells
• Rarely identified in cytology samples
• Neuroendocrine cells are situated towards the basement membrane.
• Known as Feyrter or K (Kultschitzsky) cells.
• They contain neurosecretory granules producing locally active
hormones into the blood
Clara Cells
• Line the terminal bronchioles.
➢
➢
➢
Cells are cuboidal or tall / columnar
Non ciliated
Non mucous secreting
• Cells secrete various protein that clears the airways and have the
potential to differentiate into ciliated cells.
• Cannot be recognized or identified in routine cytology samples
Alveolar Cells
Type I & II Pneumocytes
• Alveolar air sacs lined by cells known as type I and type II pneumocytes.
• Type I pneumocytes are large, thin cells stretched across a large surface
and cover the areas where gaseous exchange takes place.
• Type II pneumocytes manufacture surfactant, a surface active material
that prevents collapse of the air-sacs on expiration.
• Type II pneumocytes also act as reserve cells or progenitor cells and can
differentiate into type I pneumocytes when they need to be replaced.
• Type II pneumocytes are round, single cells with central nuclei, small
nucleoli, and vacuolated cytoplasm
Alveolar Macrophages
• Alveolar macrophages (also known as histiocytes) are macrophages found
in the pulmonary alveolus which are derived from bone marrow.
• The presence of this cells indicate that a sputum specimen is satisfactory as
they indicate that the sample comes from the deep lungs
• They are phagocytes; their role is engulfing, digesting, and removal of
inhaled particles and pathogens in the alveoli.
• They also stimulate lymphocytes and other immune cells to respond to
foreign matter.
• Alveolar macrophages vary in appearance depending on their activity.
• The cytoplasm may be vacuolated and evidence of inhaled particles
(usually carbon) may be seen as brown granules with Papanicolaou stain.
Alveolar macrophages
• Single-lying/ small clusters
• Eccentric or central single/
binucleate/ multinucleate nuclei
• Nuclear shape: round-oval-beanshaped
• Granular chromatin
• Inconspicuous nucleoli
• Cytoplasm basophilic, foamy/
finely vacuolated/ ingested
particles
Carbon-laden alveoli macrophages
Summary of different cell types in respiratory tract
Inflammatory Cells
• Mainly neutrophils and lymphocytes, are invariably present in low numbers
and are only of diagnostic significance if markedly increased.
- Acute inflammation: seen in patients with pneumonia, acute
bronchitis and alveolar abscess
- Chronic Inflammation: seen in patients with follicular bronchitis, viral
infections
• A predominance of one inflammatory cell type may however be significant,
for example, when eosinophils are conspicuous (frequently noted from
patient with asthma or those with infections due to fungi or parasites.
Non-Cellular Components
• Non-living structures may be present in specimens RT
• Some indicate specific problem; while others only serve to confuse
and produce incorrect diagnosis
• Structures may be derived from:
- patient (endogenous)
- contaminant after specimen collection (exogenous)
- Inhaled material
• Their recognition is important to avoid misinterpretation.
Curschmann’s Spiral
• Strands of mucous seen in
lumen of small bronchioles
• Conditions of excess mucous
production
• Coiled/ spiral like appearance,
dark staining center and
translucent periphery
• Associated with asthma, chronic
bronchitis, smoking, cancer
Ferruginous (Asbestos) Bodies
• Ferruginous (containing particles of
iron) or asbestos bodies are formed
when inhaled filamentous particles
(dust) become coated in protein
and iron.
• Segmented or bamboo shaped
with knobbed or bulbous ends
(dumbbell), ranging from 5 to 200
μm in length, they stain goldenyellow to black with Papanicolaou
stain and blue with Prussian blue
stain
Charcot- Leyden Crystals
• Needle-shaped, elongated,
orangeophilic/pink/yellow
structures formed from
breakdown products of
eosinophil granules.
• May be seen in patients with
severe allergic disorders like
asthma, allergy, parasitic or
fungal infections or eosinophilic
pneumonia
Corpora Amylacea
• Non-calcified small glycoprotein
of alveolar space – rounded,
concentrically laminated not
calcified with radial striations
acidophilic structures
• They stain pale pink, are Congo
red positive
• Significance unclear in the lung,
often associated with benign
conditions
Psammoma Bodies
• Concentrically laminated calcified
structures contain phosphate, iron
and magnesium
• Seen in malignant tumors that have
papillary architecture, like primary
pulmonary adenocarcinoma,
mesothelioma, and metastatic
thyroid or ovarian cancer.
• They are also seen in benign
conditions like pulmonary
tuberculosis and alveolar
microlithiasis.
Contaminants
• Any structure breathed into the
respiratory passage or any particle of
masticated food may appear on
respiratory specimens:
-Alternaria (arial or water born)forms light brown conidia with an
internal segmented structure
-Food- includes fragments of plant
tissue- characteristic rectangular
shape, thick cellulose walls and dark
nuclei
meat fibre are elongated and may
show radial striations
-Colonial growth of normal flora –
include bacteria and yeast, occur
when delay in specimen
preparation
- Pollen
• Common source of error
Contaminants
Alternaria
Meat Fibre
Vegetable cells
Specimen Collection And Sample Processing
• Cytologic specimens from the
respiratory tract are one of the
most common cytologic
specimens received by the
laboratory.
• Indications for respiratory
cytology:
• Diagnosis of malignancy (primary
and metastatic)
• Diagnosis of infection, particularly
in immunosuppressed individuals.
• Diagnosis of benign lesions.
Sampling Methods
• Sputum
• Bronchial washings
• Bronchial brushings
• Bronchoalveolar lavage
• FNA
• Transbronchial
• Transthoracic
Sputum
• Once the most frequently examined specimen from RT – easy to
obtain / little discomfort to patients
• Complex mucoid product from airways containing variety of cellular
non-cellular material produced by host or inhaled
• Microscopic examination may indicate benign or malignant conditions
of the airways
• Most specific for squamous cell carcinoma
Sputum
• The adequacy of a sputum sample is established by finding numerous
pulmonary macrophages.
• Specimens consisting merely of squamous cells, bacteria, and Candida
organisms are unsatisfactory because they represent only oral
contents.
• The presence of numerous macrophages indicates that a satisfactory,
deep cough specimen of the lower respiratory tract has been
obtained.
Method Of Collection
• Produced by deep coughing
• Early morning deep cough specimens preferred
• Patient must brush teeth/ rinse mouth – remove any exfoliated oral cells
that would contaminate the sputum sample
• Patient must inhale deeply repeatedly and exhale with an explosive cough
into sterile container
• Sputum induced – patient inhale nebulized water or saline
• If delay in specimen prep, patient must expectorate into a 70% ethanol
solution to prefix specimen
• Multiple samples collected over three days increases sensitivity of test
Lab Preparation
• Usually prepared by the “pick and smear” method
• Saccomanno Method
Sputum Preparation
“Pick and smear” method:
• Pour sputum sample into Petri dish
• Examine sample for areas of tissue fragments or bloody material
• Material is smeared between two glass slides.
• Fixed in alcohol and stained by the Papanicolaou method
Sputum Preparation
Saccomano Method:
• Samples collected in mixture of
50% ethanol and 2% carbowax
• Blending at high speeds – using
a blender
• Samples concentrated by
centrifugation and preparing the
smears from the centrifuged cell
button
Sputum
Advantages
Disadvantages
Easily, painlessly obtained, if spontaneous
Difficult if not spontaneous
Good for detection of central tumours
Poor for detection of peripheral tumours
Extensive area sampled
Cannot localise lesion
Accurate esp. squamous and small cell
carcinomas
Less accurate for adenocarcinoma
Bronchoscopy
Bronchial Aspirates and Washing
• Sampling of the lower respiratory tract occurred with the
development of the flexible fiberoptic bronchoscope in the late
1960s.
• Allows any part of RT to be sampled
• Bronchial secretions can be aspirated directly from the lower
respiratory tract through the bronchoscope, or an alternative (and
more common) method is to “wash” the mucosa by instilling 3 to 10
ml of saline and suctioning the washings into a trap.
• Fluid centrifuged , concentrate used to make smears
Brochoalveolar Lavage
• BAL the bronchoscope is wedged into position as far as it will go in
order to sample the distal airways, which are flushed with sterile
saline.
• Specimen is centrifuged and cell palate is used to make smears
• BAL is particularly useful for the diagnosis of opportunistic infections
in immunocompromised patients, TB and malignancy
(adenocarcinoma).
• The specimen can be examined cytologically and a portion also
submitted for microbiologic studies.
Fine Needle Aspiration Biopsy
• Diagnostic procedure that can be
carried out on an outpatient basis.
• A needle is passed through the
chest wall (transthoracic) or
through the bronchial wall
(transbronchial) into the
pulmonary wall.
• The contents of the needle are
expelled onto a slide and a smear is
made with a second slide by pulling
the slides apart.
• Fix or air-dry & stained in lab with
pap stain
Transbronchial (TFNA)
• Transbronchial FNA is a special modification of needle aspiration used
when the lung neoplasm has not invaded through the bronchial mucosa
and is not accessible through sputum or bronchial brushing/washing
• Used for sampling of mediastinal lymph nodes (mediastinum area between
lungs that contain heart, aorta, trachea and lymph nodes)
• TBNA is also used diagnosis of lung cancer and also staging procedure
• Procedure involves insertion of a needle a flexible through fibreoptic
bronchoscope
• Penetration of bronchial wall and aspiration of cytological material lying
beyond
Transthoracic Fine Needle Aspirations
• Performed under Computed tomography (CT) guidance
• Involves insertion of fine needle through the thoracic wall and pleura
to obtain specimen.
• Indicated for peripheral lesions
Homework
List the advantages and disadvantages of different sampling techniques
in respiratory cytology.
Feedback to be given in our next class.
Routine Staining Method
• Papanicolaou stain widely used as routine stain – results in welldefined nuclear detail and cytoplasmic transparency
• Romanowsky staining, such as May-Grunwald-Giemza or Diff-Quik
used to compliment Pap stain – allow better visualization of
cytoplasmic features and enhance cell size
Ancillary Test
Immunocytochemistry (ICC):
• Immunocytochemistry is an ancillary technique that is used in the
anatomical pathology laboratory for the detection and visualization of
proteins and peptides (antigens) in cells using biomolecules
(antibodies) capable of binding the protein of interest (antigenantibody interaction).
• ICC divided into direct and indirect methods
ICC
Direct Method
• Direct method rely on direct
visualization of antigen-antibody
reaction using primary antibody
that is conjugated with a visible
marker (probe, tag or label) –
fluorescent dye
Disadvantage
• Poor sensitivity – due low
concentration of antigenic
components might be
undetectable
ICC
Indirect method
• Secondary antibody is applied
after a primary antigen-antibody
reaction takes place, the second
antibody is tagged or several
antibodies bind to a primary
antibody
Advantage
• Amplifies the signal- increased
sensitivity
ICC
• The tag is an enzyme, which in itself is invisible. Only
when a chromogenic substrate or chromogen is
applied does the reaction become visible.
• A coloured product indicates a positive
immunoreaction between antibody and the antigen of
interest.
• Horseradish peroxidase used as tag.
• This enzyme cannot be seen directly, the addition of
hydrogen peroxide and the colourless substance 3,3’diaminobenzidene (DAB) produces a brown reaction
product which is easily recognized microscopically.
Brown ICC reaction indicates the
site of antigen-antibody reaction
Ancillary Tests
Special Stains
• Used to ID specific elements in
cytological preparations after
routine evaluation by pap and
Romanowsky stained slide
• Example:
o Periodic acid Schiff (PAS)
o Grocott’s methenamine silver
o Alcian Blue PAS
o Congo Red
• PAS- used to demonstrate
carbohydrates. Glucose
produces by cell or organism
reacts with Schiff reagent to
produce magenta colour – used
to id some pathogenic fungi and
other constituents of a cell