thinprep non-gyn lecture series - cytology · pdf filehologic proprietary © 2012...
TRANSCRIPT
![Page 1: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/1.jpg)
Hologic Proprietary © 2012
ThinPrep® Non-Gyn
Lecture Series
Respiratory Cytology
![Page 2: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/2.jpg)
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Benefits of
ThinPrep® Technology
Benefits of ThinPrep Non-Gyn for
Respiratory (Mucoid) specimens:
• Control cellular density
• Eliminate preparation artifact
• Minimize number of slides per patient
• Eliminate mucoid glycoprotein
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Required Materials
• ThinPrep® 2000 Processor or
ThinPrep 5000 Processor
• ThinPrep Microscope Slides
• ThinPrep Non-Gyn Filters (Blue)
• Multi-Mix™ Racked Vortexor
• CytoLyt® and PreservCyt® Solutions
![Page 4: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/4.jpg)
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Required Materials
• 50 ml capacity swing arm centrifuge
• 50 ml centrifuge tubes
• Slide staining system and reagents
• 1 ml plastic transfer pipettes
• 95% alcohol
• Coverslips and mounting media
Optional: Glacial acetic acid, DTT and saline for troubleshooting
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Recommended Collection
Media
• CytoLyt®
• Plasma-Lyte®
• Polysol®
• Balanced electrolyte solutions
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Non-Recommended Collection
Media
• Sacomanno and other solutions
containing carbowax
• Alcohol
• Mucollexx®
• Culture Media, RPMI Solution
• PBS
• Solutions containing formalin
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Hologic® Solutions
• CytoLyt®
• PreservCyt®
Copyright © 2012 Hologic, All rights reserved.
Copyright © 2012 Hologic, All rights reserved.
![Page 8: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/8.jpg)
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Hologic® Solutions CytoLyt® Solution
• Methanol-based, buffered preservative solution
- Lyses red blood cells
- Prevents protein precipitation
- Dissolves mucus
- Preserves morphology for 8 days at room
temperature
• Intended as transport medium
• Used in specimen preparation prior to
processing
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Hologic® Solutions PreservCyt® Solution
• Methanol based, buffered solution
• Specimens must be added to PreservCyt
Solution prior to processing
• PreservCyt Solution cannot be substituted
with any other reagents
• Cells in PreservCyt Solution are preserved
for up to 3 weeks in a temperature range
between 4 -37 C
![Page 10: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/10.jpg)
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Specimen Types
• Sputum
• Bronchial Brushing
• Bronchial Washing
• Bronchoalveolar Lavage
• Fine Needle Aspiration (FNA) Biopsy,
Transbronchial FNA, Endobronchial
Ultrasound (EBUS) FNA
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Sputum
• Easily obtained if spontaneous
• 3 to 5 consecutive day, early morning,
deep cough specimens preferred
• Can be induced if not spontaneous by
inhalation of an aerosolized solution
• Good for detection of central tumor
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Bronchial Brushing
• Collected with a small brush under
visual control using a fiberoptic
bronchoscopic instrument
• Good for detection of peripheral tumors
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Bronchial Washing
• Collected by instilling 3 to 5 mL of
balanced salt solution through the
bronchoscope and re-aspirating the
resulting material
• Good for detection of peripheral tumors
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Bronchoalveolar Lavage (BAL)
• Collected by infusing and re-aspirating a sterile
saline solution in the distal segments of the lung
using a fiberoptic bronchoscope
• Utilized in the therapy of diseases such as pulmonary
alveolar proteinosis, cystic fibrosis, pulmonary
alveolar microlithiasism and asthma
• Good for detection of peripheral cancers as well as
opportunistic infections in immunocompromised
patients
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Transbronchial FNA (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
• An endobronchial aspirate may also be preformed
under ultrasound guidance to reach these lesions,
commonly referred to as Endobronchial Ultrasound
(EBUS)
• Good for detection of lesions that are not visible
during conventional bronchoscopy
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Percutaneous FNA Biopsy
• A fine needle attached to a syringe is passed
through the chest wall into the pulmonary mass
visualized either by fluoroscopy, computed
tomography, or ultrasound
• Good as an alternative to surgery for detection
of pulmonary lesions that are not accessible
during conventional bronchoscopy
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Sample Collection Mucoid Specimens
• Sputum: Collect directly into 30 ml
CytoLyt® solution
• Bronchial Brushing: Deposit collection
brush directly into a 30 ml prefilled
CytoLyt solution tube
• Bronchial Washing/Lavage: Collect
with a balanced electrolyte solution
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Sample Collection Fine Needle Aspirates
• Deposit and rinse the entire sample
into a centrifuge tube containing 30
ml of CytoLyt® solution or a balanced
electrolyte solution, such as Polysol®
or Plasma-Lyte®
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Sample Preparation Mucoid Specimens
1. Sample Collection
Optional: If DTT is being used, add stock before agitation.
See next slide for stock preparation.
2. Mechanical agitation
3. Concentrate by centrifugation
4. Pour off supernatant and vortex to re-suspend cell pellet
5. Evaluate cell pellet
If cell pellet is not in liquid form do a CytoLyt® wash and
repeat steps 2-4
6. Add recommended # of drops of specimen to PreservCyt®
Solution Vial
7. Allow to stand for 15 minutes
8. Run on ThinPrep® 2000 using Sequence 3 or ThinPrep 5000
using Sequence Non-Gyn
9. Fix, Stain, and Evaluate
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Sample Preparation DTT Protocol for Mucoid Specimens
Preparing a DTT Stock Solution:
• Add 2.5g DTT to 30 ml of CytoLyt®
Solution
• This solution is suitable for use for 1 week
when stored at room temperature (15-
30 C)
• Add 1 ml of stock solution to the sample
prior to vortexing
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Sample Preparation DTT Protocol for Mucoid Specimens
Preparing a DTT Single Use Solution:
• Add 0.08g DTT in individual 50 ml conical
tubes. Place in freezer
• When ready for use, add 30 ml of CytoLyt®
Solution and shake/vortex to dissolve
• Add specimen to DTT
• Vortex and continue processing protocol
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Sample Preparation Fine Needle Aspiration
1. Collection
2. Concentrate by centrifugation - 600g for 10 minutes
3. Pour off supernatant and vortex to re-suspend cell pellet
4. Evaluate cell pellet
• If cell pellet is not free of blood, add 30 ml of CytoLyt®
Solution to cell pellet and repeat from step 2
5. Add recommended # of drops of specimen to PreservCyt®
Solution Vial
6. Allow to stand for 15 minutes
7. Run on ThinPrep® 2000 Processor using Sequence 3 or
ThinPrep 5000 using Sequence Non-Gyn
8. Fix, Stain, and Evaluate
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Sample Preparation Protocol
Summary
• ThinPrep® Non-Gyn Respiratory
Protocol
- Sputum
- Bronchial Washing
- Bronchial Brushing
- Bronchoalveolar Lavage
• ThinPrep Non-Gyn FNA Protocol
- FNA, Transbronchial FNA, EBUS
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Sample Preparation
Techniques
• Mechanical Agitation
- Mucoid specimens require vigorous
agitation in CytoLyt® solution to break up the
mucus.
• Two recommended methods:
- Vortex for 5 minutes
- Blend for a few seconds
Note: Agitation times for both methods may vary
depending on specimen consistency
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Sample Preparation
Techniques
• Centrifugation - 600g for 10 minutes or
1200g for 5 minutes
- Concentrates the cellular material in order
to separate the cellular components from the
supernatant
Refer to Centrifuge Speed Chart in the ThinPrep® 2000 or
ThinPrep 5000 Processor Manual, Non-Gynecologic section, to
determine the correct speed for your centrifuge to obtain force of
600g or 1200g
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Sample Preparation
Techniques
• Pour off supernatant
- Invert the centrifuge tube 180 in one
smooth movement, pour off all supernatant
and return tube to its original position
(Note: Failure to completely pour off the supernatant
may result in a sparsely cellular sample due to
dilution of the cell pellet).
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Sample Preparation
Techniques
• Vortex to re-suspend cell pellet
- Randomizes the cell pellet and improves
the results of the CytoLyt® solution washing
procedure
- Place the centrifuge tube onto a vortexor
and agitate the cell pellet for 3 seconds or
vortex manually by syringing the pellet back
and forth with a plastic pipette
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Sample Preparation
Techniques
• CytoLyt® Solution Wash
- Preserve cellular morphology while lysing
red blood cells, dissolving mucus and
reducing protein precipitation
- Add 30 ml of CytoLyt Solution to a cell
pellet, vortex for 5 minutes (mucoid
specimens only), concentrate by
centrifugation, pour off the supernatant and
vortex to resuspend the cell pellet
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Sample Preparation
Techniques
• Evaluate cell pellet
- If cell pellet is white, pale pink, tan or not
visible add specimen to PreservCyt®
Solution vial (# of drops added is
dependant on sample volume and will be
discussed on future slides)
- If cell pellet is distinctly red or brown
indicating the presence of blood conduct
a CytoLyt® wash
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Sample Preparation
Techniques
• Evaluate cell pellet
- To test for liquid form, draw a small amount
of sample into a pipette and deliver drops
back into the tube.
- If the drops appear stringy or gelatinous,
the mucus must be further liquefied.
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Sample Preparation
Techniques
• Calculate how many drops of specimen
to add to PreservCyt® vial:
- If pellet is clearly visible and the pellet
volume is ≤ 1ml (if not consider the next 2
slides)
• Vortex pellet and transfer 2 drops to a
fresh PreservCyt Solution vial
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Sample Preparation
Techniques
• Calculate how many drops of specimen
to add to PreservCyt® vial:
- If pellet volume is ≥1ml • Add 1ml of CytoLyt® Solution into the tube
and vortex briefly to resuspend the cell pellet
• Transfer 1 drop of the specimen to a fresh
PreservCyt Solution vial
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Sample Preparation
Techniques
• Calculate how many drops of specimen
to add to PreservCyt® vial:
- If pellet is not visible or scant • Add contents of a fresh PreservCyt Solution
vial into the tube and vortex briefly to mix the
solution
• Pour entire sample back into the vial
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Sample Preparation
Troubleshooting
• Due to the biological variability among
samples and variability in collection
methods, standard processing may yield
a slide that indicates further
troubleshooting may be needed.
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Sample Preparation
Troubleshooting
• After staining, you may observe the
following irregularities: • Non-uniform distribution of cells in the cell spot
without a “sample is dilute” message
• Uneven distribution in the form of a ring or halo
of cellular material and/or white blood cells
• A sparse cell spot lacking in cellular component
and containing blood, protein and debris – may
be accompanied by a “sample is dilute” message
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Techniques Used in
Troubleshooting
• Diluting the Sample 20 to 1
• Glacial Acetic Acid Wash for Blood and Non-
Cellular Debris
• Saline Wash for Protein
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Techniques Used in
Troubleshooting
• Diluting the Sample 20 to 1
- Add 1ml of the sample that is suspended in
PreservCyt® Solution to a new PreservCyt
Solution vial (20ml). This is most accurately
done with a calibrated pipette.
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Techniques Used in
Troubleshooting
• Glacial Acetic Acid Wash for Blood and Non-
Cellular Debris
- If sample is bloody, it can be further
washed using a solution of 9 parts CytoLyt®
Solution and 1 part Glacial Acetic acid.
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Techniques Used in
Troubleshooting
• Saline Wash for Protein
- If sample contains protein, it can be further
washed with saline solution in place of
CytoLyt® Solution.
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Troubleshooting
Mucoid Specimens
“Sample is Dilute”
message
No, continue to
next slide
Yes
Check to see if
cellularity is
adequate. If not, use
more of the pellet, if
available and prepare
new slide.
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Troubleshooting
Mucoid Specimens
Does the slide have a
“halo” of cellular
material and/or white
blood cells?
No, continue to next
slide
Yes
Dilute the sample
20:1 by adding 1ml of
sample to a new
PreservCyt® Solution
vial. Prepare new
slide.
If halo is present on
the new slide, contact
Hologic® Technical
Service.
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Troubleshooting
Mucoid Specimens
Is the slide sparse and
does it contain
mucus?
No
Contact Hologic®
Technical Service
Yes
Centrifuge remaining
specimen from PreservCyt®
vial, pour off and vortex.
Perform CytoLyt® wash and
evaluate cell pellet
appearance. If pellet contains
mucus repeat CytoLyt wash.
Add to PreservCyt vial and
prepare new slide.
If resulting slide is sparse, contact
Hologic Technical Service.
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Troubleshooting
Bloody or Proteinaceous Specimens
“Sample is Dilute”
message
No, continue to
next slide
Yes
Check to see if
cellularity is
adequate. If not, use
more of the pellet, if
available and
prepare new slide.
![Page 44: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/44.jpg)
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Troubleshooting
Bloody or Proteinaceous Specimens
Does the slide have a
“halo” of cellular
material and/or white
blood cells?
No, continue to next
slide
Yes
Dilute the sample 20:1
by adding 1ml of
residual sample to a
new PreservCyt®
Solution vial and
prepare new slide.
If halo is present on the
new slide, contact Hologic®
Technical Service.
![Page 45: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/45.jpg)
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Troubleshooting
Bloody or Proteinaceous Specimens
Is the slide sparse and does
it contain blood, protein or
non-cellular debris?
Yes-protein
Centrifuge remaining specimen from
PreservCyt vial, pour off. Add 30 ml of
saline to sample, centrifuge, pour off
and vortex. Add to PreservCyt vial and
prepare new slide. If resulting slide is
sparse, contact Hologic Technical
Service.
No
Contact Hologic®
Technical Service
Yes-blood
or non-
cellular
debris
Centrifuge remaining specimen
from PreservCyt® vial, pour off.
Add 30ml of a 9:1 CytoLyt® to
glacial acetic acid solution to
the sample, centrifuge, pour off
and vortex. Add to PreservCyt
vial and prepare new slide. If
the resulting slide is sparse,
contact Hologic Technical
Service.
![Page 46: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/46.jpg)
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Troubleshooting
Common Artifacts
• Smudged Nuclear Detail
• Compression Artifact
• Staining Artifact
• Edge of the Cylinder Artifact
![Page 47: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/47.jpg)
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Troubleshooting
Common Artifacts
• Smudged Nuclear Detail
• May occur if specimen is collected in saline,
PBS or RPMI
• To avoid this, collect the sample either
fresh, in CytoLyt® or in PreservCyt® solution
![Page 48: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/48.jpg)
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Troubleshooting
Common Artifacts
• Compression Artifact
• Appears as “air dry” artifact on the perimeter
of the cell spot
• Due to the compression of cells between
the edge of the filter and the glass of the slide
![Page 49: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/49.jpg)
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Troubleshooting
Common Artifacts
• Staining Artifact
• Mimics air-drying
• Appears as a red or orange central staining
primarily in cell clusters or groups
• Due to the incomplete rinsing of counterstains.
• To eliminate this artifact, fresh alcohol baths
or an additional rinse step after the
cytoplasmic stains is required
![Page 50: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/50.jpg)
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Troubleshooting
Common Artifacts
• Edge of the Cylinder Artifact
• Narrow rim of cellular material just beyond
the circumference of the cell spot
• Result of cells from the outer edge of the
wet filter cylinder being transferred to
the glass slide
![Page 51: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/51.jpg)
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Anatomy
![Page 52: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/52.jpg)
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Histology of the Epithelium
Other Cell Types Include: • Goblet cells • Clara cells • Kulchitsky cells • Pneumocytes
Respiratory system is lined by two types of
epithelium:
• Non-Cornified Stratified Squamous Epithelium
• Pseudostratified Ciliated Columnar Epithelium
Courtesy of Brian Bich at Lake Superior College
![Page 53: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/53.jpg)
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Specimen Adequacy
• Sputum- presence of numerous alveolar
macrophages
• Bronchial Brushing- presence of
numerous bronchial cells
• BAL/Bronchial Washing- presence of
bronchial cells and alveolar
macrophages
• FNA- presence of diagnostic material
![Page 54: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/54.jpg)
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Normal Components and
Findings
• Ciliated columnar epithelial cells
Nuclei:
- Basally oriented
- Round to oval with smooth nuclear membranes
- May contain a nucleolus
- Fine to mildly coarse chromatin
- Variable in size
Cytoplasm:
- Homogenous and basophilic
![Page 55: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/55.jpg)
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40x
Ciliated Columnar Epithelial Cells
Copyright © 2012 Hologic, All rights reserved.
![Page 56: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/56.jpg)
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Normal Components and Findings
• Goblet Cells
- Commonly seen in bronchial washing and
brushing specimens
- Often in clusters or sheets
- Usually one for every 5-10 ciliated
epithelial cell
- Abundant finely vacuolated cytoplasm
filled with mucus
![Page 57: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/57.jpg)
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Goblet Cells
60x Copyright © 2012 Hologic, All rights reserved.
![Page 58: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/58.jpg)
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• Squamous cells
- More common in sputum than in
washings/brushings
- Similar to those seen in Gyn samples
- Predominantly superficial
- Anucleated squames, intermediate cells and
benign squamous pearls may also be present
Normal Components and Findings
![Page 59: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/59.jpg)
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Normal Components and Findings
• Squamous Metaplasia
- Similar to metaplasia seen in Gyn
samples
- Uniform in size and shape
- Appear in loose sheets, in a cobblestone
arrangement
- Nuclei are round
- Chromatin ranges from granular to coarse
or pyknotic
- No nucleoli, unless cells are reactive
![Page 60: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/60.jpg)
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Squamous Metaplasia
60x 60x Copyright © 2012 Hologic, All rights reserved Copyright © 2012 Hologic, All rights reserved
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Normal Components and Findings
• Clara cells
- Non-ciliated bronchiolar cells
- Secrete a protein that acts as a clarificant,
a function similar to mucus
• Kulchitsky cells
- Scattered basal epithelial cells
- Contain neurosecretory granules
- Parent cells of carcinoid tumors
![Page 62: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/62.jpg)
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Normal Components and Findings
• Reserve cells
- Small, round, lymphocyte-like cells
- Nuclei are centrally located, uniform and
hyperchromatic
- Can only be specifically identified in clusters
• Pneumocytes
- Type I Pneumocytes
- Type II Pneumocytes: (Bronchoalveolar cells)
![Page 63: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/63.jpg)
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Reserve cells
Copyright © 2012 Hologic, All rights reserved Copyright © 2012 Hologic, All rights reserved
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• Pulmonary macrophages
- Cells may appear singly or in clusters
- Nuclei may be round, oval, reniform or
other shape
- Multinucleation may be present
- Cytoplasm is foamy and may be
abundant
- May contain ingested debris
Normal Components and Findings
![Page 65: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/65.jpg)
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Macrophages
Carbon-laden Hemosiderin-laden
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Macrophages
Lipophages
Copyright © 2012 Hologic, All rights reserved
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Normal Components and Findings
• Ciliocytophthoria
- Small ciliated tufts
• Inflammatory cells
- PMN’s
- Lymphocytes
- Plasma cells
- Eosinophils
![Page 68: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/68.jpg)
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Ciliocytophthoria
Copyright © 2012 Hologic, All rights reserved
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Normal Components and Findings
• Bacteria/Fungus
• Actinomyces
• M. tuberculosis
• Aspergillus spp.
• Candida spp.
• Pneumocystis
carinii
• Cryptococcus
• Histoplasma
• Blastomyces
• Coccidioides
• Zygomyces
Infectious agents that may be seen:
![Page 70: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/70.jpg)
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Actinomyces
60x Copyright © 2012 Hologic, All rights reserved
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Tuberculosis
• Caused by infection with Mycobacterium
tuberculosis and commonly results in
granulomatous inflammation
Cytologic features:
- Aggregates of epithelioid histiocytes,
lymphocytes, and Langhans giant cells
• Necrosis may or may not be present
• Definitive diagnosis can be made with help
from special stains or microbiologic culture
![Page 72: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/72.jpg)
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Aspergillus spp.
40x 40x Copyright © 2012 Hologic, All rights reserved Copyright © 2012 Hologic, All rights reserved
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Candida spp.
40x Copyright © 2012 Hologic, All rights reserved
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Pneumocystis carinii
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Cryptococcus
Copyright © 2012 Hologic, All rights reserved Copyright © 2012 Hologic, All rights reserved
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Histoplasma
• Contracted by inhalation of spores of
Histoplasma capsulatum
• More commonly affects patients who
are immunocompromised
• Organism often presents within
cytoplasm of macrophages
• Is very small and may be overlooked if
silver stains are not used
![Page 77: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/77.jpg)
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Blastomyces
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Coccidioides
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Zygomyces
40x Copyright © 2012 Hologic, All rights reserved
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Normal Components and Findings
• Viral
• Herpes
• CMV
• Adenovirus
• Measles
• Respiratory
Syncytial Virus
(RSV)
• Nematode
• Strongyloides
Infectious agents that may be seen:
![Page 81: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/81.jpg)
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Herpes (HSV)
60x 60x Copyright © 2012 Hologic, All rights reserved Copyright © 2012 Hologic, All rights reserved
![Page 82: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/82.jpg)
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Cytomegalovirus (CMV)
60x Copyright © 2012 Hologic, All rights reserved
![Page 83: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/83.jpg)
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Adenovirus
• Usually causes minor illness but adenovirus
pneumonia can be severe and fatal, especially for
those who are immunocompromised
• Causes two types of nuclear inclusions
- “The smudge cell”- large basophilic inclusions fill
the entire nucleus and obscure chromatin detail
- Eosinophilic inclusion that resembles the Cowdry A
inclusion of herpes simplex virus
• Ciliocytophthoria can be prominent
![Page 84: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/84.jpg)
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Measles and Respiratory
Syncytial Virus (RSV)
• Measles is highly contagious
• Caused by the rubeola virus
• Incidence limited because of vaccination
• Measles pneumonia occurs as an opportunistic
complication in children who are
immunocompromised
Cytologic features:
- Enormous multinucleated cells with cytoplasmic and
nuclear inclusions
• RSV has similar findings and is usually confirmed by
detecting RSV antigen in BAL specimens
![Page 85: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/85.jpg)
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Strongyloides
20x Copyright © 2012 Hologic, All rights reserved
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Normal Components and Findings
Acellular Material:
• Curshmann’s spirals
• Amorphous mucus
• Corpora Amylacea
• Amyloid
• Psammoma bodies
• Charcot-Leyden Crystals
Other Findings/Contaminants:
• Ferruginous bodies
• Undigested food particles
• Plant material
• Vegetable cells
• Pollen
![Page 87: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/87.jpg)
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Curshmann’s spirals
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Amorphous Mucus
• Also known as mucus bodies or blue
blobs
• Can mimic naked malignant nuclei,
lacking distinct chromatin pattern or
cytoplasm
• Can be round or ring shaped
![Page 89: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/89.jpg)
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Corpora Amylacea
• Spherical structures with circumferential
and radiating lines
• Measure between 30 and 200 µm
• Indistinguishable from those seen in the
prostate
• No known clinical significance
• More commonly seen in older patients
![Page 90: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/90.jpg)
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Amyloid
• Occurs as multiple, irregular, dense, acellular
fragments of eosinophilic material
• Waxy, homogenous appearance, with sharp
often scalloped margins
• Characteristic apple green birefringence is
seen under polarized light with Congo red
staining
![Page 91: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/91.jpg)
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Psammoma Bodies
• Concentrically laminated, calcified, basophilic
bodies surrounded by cells
• May be associated with cancers such as
bronchioloalveolar carcinoma or metastatic
papillary carcinoma
• May also be seen in benign conditions such
as alveolar microlithiasis
![Page 92: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/92.jpg)
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Charcot-Leyden Crystals
• Rhomboid-shaped, orangeophilic
structures derived from degenerating
eosinophils
• Can be seen in patients with severe
allergic disorders like asthma
![Page 93: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/93.jpg)
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Ferruginous bodies
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Undigested Food/Plant Material
• Food particles are common in sputum
• Meat is characterized by cross striations
• Plant cells may have very dark, smudgy
nuclei with translucent refractile cell walls
• May mimic adenocarcinoma or squamous
cell carcinoma
![Page 95: ThinPrep Non-Gyn Lecture Series - Cytology · PDF fileHologic Proprietary © 2012 ThinPrep® Non-Gyn Lecture Series Respiratory Cytology](https://reader031.vdocument.in/reader031/viewer/2022012305/5a7923017f8b9a7b548d701f/html5/thumbnails/95.jpg)
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Vegetable cell
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Pollen
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Benign Entities and Changes
• Reactive changes are common and
may be due to:
- Instrumentation
- Infection or toxins (including
smoking or tracheobronchial
disease)
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Reactive Changes
• Features of reactive bronchial cells
may include:
- Enlarged pleomorphic nuclei
- Prominent nucleoli
- Coarse, hyperchromatic chromatin
- Multinucleation
- Abundant cytoplasm
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Reparative Changes
• Similar to that seen in Gyn samples
• Atypia can range from mild to severe,
and may mimic cancer
• Unlike with cancer, both the nucleus
and cytoplasm of reparative cells
increase in size, maintaining a benign
N/C ratio
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Benign Proliferation
The bronchial epithelium can undergo
a series of changes due to
chronic irritation:
• Reserve Cell Hyperplasia
• Squamous Metaplasia
• Parakeratosis, Atypical Parakeratosis
• Bronchial Hyperplasia
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Benign Proliferation
Reserve Cell Hyperplasia (RCH)
- Tightly cohesive groups of reserve
cells
- Nuclear molding may identified
- Differential diagnoses include
lymphocytes and small cell carcinoma
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Reserve Cell Hyperplasia
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Benign Proliferation
Squamous Metaplasia
- Very common, occurring in 20% to 80% of
all patients
- May be present due to irritation but may
also be associated with cancer
- Frequently occurs with RCH
- Immature respiratory metaplasia is smaller
with more polygonal cytoplasm
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Squamous Metaplasia
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Benign Proliferation
Parakeratosis/Atypical Parakeratosis
- Similar to parakeratosis seen in Gyn
samples
- Associated with irritation/inflammation or
dysplasia/cancer
- Usually arises in the mouth or in the
tracheobronchial tree
- May be difficult to distinguish from
keratinizing squamous cell carcinoma
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Benign Proliferation
Bronchial Hyperplasia
- Pseudopapillary, 3-dimensional groups
of reactive/atypical bronchial cells
- Reactive nuclei are uniform in size and shape
- Fine to coarse, evenly distributed chromatin
- Nucleoli are uniform but prominent
- Cytoplasm is variable and may have
vacuoles of varying sizes
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Pneumoconioses
Pneumoconiosis
- name given to the occupational and restrictive
lung disease caused by inhalation of dust
- the type of dust gives each disease its name
• Silicosis (silica)
• Asbestosis (asbestos)
• Berylliosis (beryllium)
• Anthracosis (coal, carbon)
• Byssinosis (cotton)
• Hemosiderosis (iron)
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Miscellaneous Conditions
• Eosinophilic Pneumonia- presence of
marked increase in eosinophils
-Also known as Löffler’s Pneumonia
• Giant Cell Interstitial Pneumonia- often
caused by exposure to hard metals
- Benign multinucleated giant cell histiocytes
which may contain phagocytosed debris or
cells
• Atelectasis- collapse of all or part of lung
- Cytology presence with atypical squamous
cells and foreign body macrophages
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Noninfectious Diseases
• Sarcoidosis-common disease,
characterized by noncaseating
granulomas in many organs, commonly
the lung
Cytologic features:
- Aggregates of epithelioid histiocytes
- Multinucleated giant cells
- Lymphocytes
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Sarcoidosis
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Noninfectious Diseases
Continued
• Wegener Granulomatosis- presents as
a lung mass with or without involvement
of other organs
Cytologic features: non specific
- Necrotic collagen, giant cells,
granulomas, and neutrophils
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Benign Neoplasms
• Pulmonary Hamartoma
• Inflammatory Myofibroblastic Tumor
(IMT)
• Endobronchial Granular Cell Tumor
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Pulmonary Hamartoma
Cytologic features:
- Benign glandular cells
- Immature fibromyxoid matrix and
bland spindle cells
- Mature cartilage with chondrocytes
- Adipocytes
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Inflammatory Myofibroblastic Tumor
(IMT)
Cytologic features:
- Spindle cells
- Arranged in fascicles or storiform
pattern
- Abundant polymorphous inflammatory
cells
- Minimal if any necrosis
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Endobronchial Granular Cell Tumor
Cytologic features:
- Small clusters of macrophage-like
cells
- Nuclei are small, uniform and round to
oval
- Abundant granular cytoplasm
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Abnormal Findings
• Squamous Cell Carcinoma
• Adenocarcinoma
• Large Cell Undifferentiated
Carcinoma
• Small Cell Undifferentiated
Carcinoma
• Metastatic Carcinomas
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Squamous Cell Carcinoma
• Most common sub-type
• Range from poorly to
well differentiated
• Tumors tend to arise
centrally
• Can be diagnosed with
sputum cytology and
bronchial
brushing/washing
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Keratinizing Squamous Cell
Carcinoma
Cytologic features:
- Bizarre cell shapes
- Numerous single cells
- Nuclei are pleomorphic
- Chromatin ranges from coarse and dark to
pyknotic or ink dot-like
- Cytoplasm is dense, waxy, or “hard” with well
defined cell borders, ranges from scant to
abundant
- Extensive necrosis
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Non-Keratinizing Squamous Cell
Carcinoma
Cytologic features:
- Sheets and single cells, bizarre forms usually
absent
- Nuclei are enlarged and hyperchromatic
- Chromatin is coarse and irregular but more open
than Keratinizing Squamous Cell Carcinoma
- Nucleoli are usually prominent
- Cytoplasm is dense and often cyanophilic with
distinct cell borders
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Adenocarcinoma
• Tumors tend to arise in
the periphery of the lungs
• Two Types:
- Bronchogenic
Adenocarcinoma
- Bronchioloalveolar
Carcinoma (BAC)
• Detected more readily
by bronchial cytology
than by sputum
cytology
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Bronchogenic Adenocarcinoma
• Up to 30% of lung tumors
• 50% of all lung tumors in females
• Not always associated with smoking
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Bronchogenic Adenocarcinoma
Cytologic features:
- Papillary, cell balls or acinar structures
- Nuclei are enlarged with irregular nuclear
borders
- Chromatin ranges from fine to coarse
- Prominent macronucleoli
- Cytoplasm may range from homogenous to
foamy
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Bronchioloalveolar Carcinoma
• Peripheral lesion, rare in pure form
• 1% of lung tumors
• May arise from different cell types, including
ciliated terminal bronchiolar cells, Clara cells,
or type II alveolar pneumocytes
• More often well-differentiated
• Two types: mucinous or non-mucinous
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Bronchioloalveolar Carcinoma
Cytologic features:
- Sheets, cell balls with hobnail outlines, papillary
groups, acini and single cells
- Nucleoli may be multiple, inconspicuous or prominent
- Chromatin is pale and fine but can be moderately
hyperchromatic
- Cytoplasm varies from scant to abundant and finely
granular to clear
- Mucus may be present in background
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Pulmonary Neuroendocrine
Neoplasms
• Divided by the World Health
Organization into four distinct categories
- Typical Carcinoid
- Atypical Carcinoid
- Large Cell Undifferentiated Carcinoma
- Small Cell Undifferentiated Carcinoma
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Typical Carcinoid
• Accounts for 2-3% of all pulmonary tumors
• Low metastatic rate, 5-year survival: 90-98%
Cytologic features:
- Loosely cohesive groups and single cells
- Cells are round, oval or elongated
- Nuclei are uniform with “salt and pepper” chromatin
- Inconspicuous nucleoli
- Moderate to abundant granular cytoplasm
- Mitoses is uncommon
- Necrosis is absent
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Atypical Carcinoid
• More aggressive than Typical Carcinoid
• 5-year survival rate: 61-73%
• Cells and architecture similar to Typical
Carcinoid
Cytologic differences from Typical Carcinoid:
- Focal necrosis
- Mitoses are moderate in number
- Prominent nucleoli
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Large Cell Undifferentiated
Carcinoma
• Non-small cell tumor,
no definite differentiation
• Readily exfoliates
• May have squamous or
glandular features, or
both (commonly
demonstrable by using
electron microscope or
immunocytochemistry)
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Large Cell Undifferentiated
Carcinoma Cytologic features:
- Large malignant cells ranging from fairly uniform
to bizarre
- Nuclei are large and round to pleomorphic with
irregular to lobulated membranes
- Chromatin is fine to coarse and irregularly distributed
- Nucleoli can be prominent, multiple and irregular
- Cytoplasm is abundant and varies from delicate
to dense to granular
- Diathesis is usually present
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Small Cell Undifferentiated
Carcinoma
• Aggressive tumor
• Arises in the major
bronchi
• Central tumor
• Derived from or
mimics Kulchitsky
cells
• Capable of
producing a variety of
hormones
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Small Cell Undifferentiated
Carcinoma
Cytologic features:
- Small, 1-4x size of lymphocytes
- Nuclear molding is prominent
- Nuclei are hyperchromatic to pyknotic
- Nucleoli are inconspicuous
- Cytoplasm is scant and delicate
- Presence of diathesis and crush artifact
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Uncommon Pulmonary Tumors
• Adenoid Cystic Carcinoma
• Mucoepidermoid Carcinoma
• Clear Cell Tumor (“Sugar Tumor”)
• Sarcomas
• Lymphomas/Leukemias
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Adenoid Cystic Carcinoma
• Account for 20-30% of all cancers in the trachea but
also occur in the bronchi
• Symptoms include: cough, dyspnea and hemoptysis
• Poor long-term prognosis
• Most often diagnosed by bronchial brushings or
transbronchial needle aspiration
Cytologic features:
- Cylinders or spheres of small, benign-appearing
epithelial cells that surround basal lamina material
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Mucoepidermoid Carcinoma
• Uncommon, representing only 0.2% of lung tumors
• Develop in persons of all ages, including children
• A recurrent and tumor specific genetic aberration is
present: t (11; 19), forming the MECT1-MAML2
fusion transcription
Cytologic features:
- Presence of squamous, intermediate and mucinous
cells
- High-grade tumors show marker nuclear atypia
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Clear Cell Tumor (“Sugar Tumor”)
• Extremely rare, can occur in persons of all ages
• Typically presents as a peripheral mass ranging from
1 to 7 cm in greatest diameter
Cytologic features:
- Bland polygonal and spindle-shaped cells with a
central oval or elongated nucleus
- Cytoplasm is clear and glycogenated
• Differentials include: clear cell variant of
adenocarcinoma or Squamous Cell of the lung,
granular cell tumor, and metastatic tumors with clear-
cell features
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Sarcomas
• Primary malignant mesenchymal tumors of the lung
are rare
• Occur in adults of either sex
• One of the most common primary sarcomas is
leiomyosarcoma
• Can be large masses or small endobronchial lesions
Cytologic features:
- Sheet-like cellular aggregates
- Malignant spindle cells are single and highly
atypical
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Lymphomas
• Primary Hodgkin and non-Hodgkin lymphomas can
occur but metastatic lymphomas are more common
• Primary pulmonary non-Hodgkin lymphoma
represents less than 10% of all extranodal
lymphomas
• Most common primary non-Hodgkin lymphoma of the
lung (70-90%) is MALT lymphoma, followed by
diffuse large B-cell lymphoma
• Primary pulmonary Hodgkin lymphoma is
exceptionally rare
• Cytologic features of Hodgkin and non-Hodgkin
lymphomas are similar to those seen in other body
sites
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Leukemias
• 10% of diffuse pulmonary infiltrates in patients with
leukemia result from leukemic involvement of the
lung
• Leukemic involvement of the lung may present as a
mass
Cytologic features:
- Cells are dispersed as isolated cells, as with
lymphomas, and cellular monomorphism is
characteristic
• BAL is useful for demonstrating leukemic
involvement, especially when biopsy is
contraindicated because of coagulopathy
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Metastatic Disease
• Three times more common than primary
Adenocarcinoma of the lung
• Most common sites of origin are GI, Breast,
Lymphoma/Leukemia, Melanoma and
Sarcoma
• Multiple nodules favor metastatic disease
• In addition to clinical history, cytomorphologic
features and immunochemistry studies may
help determine primary tumor site
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Trademark Statement
CytoLyt, Hologic, PreservCyt, ThinPrep, and
UroCyte are registered trademarks of
Hologic, Inc. and/or its subsidiaries in the
United States. CytoLyt, Hologic, PreservCyt,
ThinPrep, UroCyte and associated logos are
trademarks of Hologic, Inc. and/or its
subsidiaries in other countries.
All other trademarks are the property of their
respective owners.
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For more information…
• Visit our websites: www.hologic.com, www.thinprep.com, www.cytologystuff.com
- Product Catalog
- Contact Information
- Complete Gynecologic and Non-gynecologic Bibliographies
- Cytology Case Presentations and Unknowns
- Slide Library Request Form
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Bibliography
ThinPrep® 2000 Processor Operator’s Manual
ThinPrep 5000 Processor Operator’s Manual
DeMay, Richard M.: Arts & Science of Cytopathology Exfoliative Cytology. 1996: 207-235.
DeMay, Richard M.: Practical Principles of Cytopathology.1999: 27-47.
Cibas, E., Ducatman, B.: Cytology: Diagnostic Principles and Clinical Correlates. 3rd edition, 2009: 72-98.
Keebler, C., Somrak, T. The Manual of Cytotechnology. 7th edition, 1993:161-172.
McKee, Grace T: Cytopathology. 1997: 234-254.
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Bibliography
Images:
Bich, Brian. Bronchus. 2008. Photograph. Lake Superior College, Duluth, MN. Web. Lake Superior College.
[http://employee.lsc.edu/faculty/BrianBich/Picture%20Library/Forms/AllItems.aspx?RootFolder=%2Ffaculty%2FBrianBich%2FPicture%20Library%2FAnat%2DPhys%20II%20%28Biol%201141%29%2FRespiratory%20System%20Tissues%2FBronchi%20and%20Bronchioles%20%2D%20Tutorial&View=%7b997D7559-348B-48F0-A9FE-
07741C9C8B65%7d] 3 Feb 2012.
SmartDraw® (Standard Edition) [Software]. (2011). Retrieved from http://www.smartdraw.com
ThinPrep® Non-Gyn Morphology Reference Atlas: Respiratory: 55-111.
ADS-00621 Rev. 001