®
ThinPrep Non-Gyn
Lecture Series
Thyroid Cytology
Hologic Proprietary © 2013
Benefits of
®
ThinPrep Technology
The use of ThinPrep Non-Gyn for Fine Needle
Aspiration specimens from the Thyroid:
• Optimizes cell preservation
• Standardizes specimen preparation
• Simplifies slide screening
• Minimize number of slides per patient
• Offers the versatility to perform ancillary
testing
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
Recommended Collection
Media
• CytoLyt®
• Balanced electrolyte solutions; such as
-Plasma-Lyte®
-Polysol®
Hologic Proprietary © 2013
Non-Recommended Collection
Media
• Sacomanno and other solutions
containing carbowax
• Alcohol
• Mucollexx®
• Culture Media, RPMI Solution
• PBS
• Solutions containing formalin
Hologic Proprietary © 2013
Hologic® Solutions
• CytoLyt®
• PreservCyt®
Copyright © 2012 Hologic, All rights reserved.
Copyright © 2013Hologic, All rights reserved.
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
FNA Biopsy
• Performed by a cytopathologist or clinician.
• A 23 gauge or 25 gauge needle with a 10ml
syringe is used
• After the area is cleaned with 95% ethanol, the
nodule is palpated and held in place with the
index and middle fingers
• The needle is passed thru the skin and into the
nodule. Several strokes are made with or
without vacuum created by the syringe plunger
Hologic Proprietary © 2013
Sample Collection
• Optimal: Deposit and rinse the entire sample into a
centrifuge tube containing 30 ml of CytoLyt® solution
• Secondary method: Collect into a balanced
electrolyte solution, such as Polysol® or PlasmaLyte® injection solutions
• If direct or air dried slides are desired, prepare prior
to rinsing the needle
Note: If possible, flush the needle and syringe with a sterile
anticoagulant solution prior to aspirating the sample. Some
anticoagulants may interfere with other cell processing techniques, so
use caution if you plan to use the specimen for other testing.
Hologic Proprietary © 2013
Sample Preparation
1.
2.
3.
4.
5.
6.
7.
8.
Collection
Concentrate by centrifugation - 600g for 10 minutes
Pour off supernatant and vortex to re-suspend cell pellet
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
Add recommended # of drops of specimen to PreservCyt®
Solution Vial
Allow to stand for 15 minutes
Run on ThinPrep® 2000 Processor using Sequence 3 or
ThinPrep 5000 using Sequence Non-Gyn
Fix, Stain, and Evaluate
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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).
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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 cell
pellet, concentrate by centrifugation, pour
off the supernatant and vortex to
resuspend the cell pellet
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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.
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
Techniques Used in
Troubleshooting
• Diluting the Sample 20 to 1
• Glacial Acetic Acid Wash for Blood and NonCellular Debris
• Saline Wash for Protein
Hologic Proprietary © 2013
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.
Hologic Proprietary © 2013
Techniques Used in
Troubleshooting
• Glacial Acetic Acid Wash for Blood and NonCellular Debris
- If sample is bloody, it can be further
washed using a solution of 9 parts CytoLyt®
Solution and 1 part Glacial Acetic acid.
Hologic Proprietary © 2013
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.
Hologic Proprietary © 2013
Troubleshooting
Bloody or Proteinaceous Specimens
“Sample is Dilute”
message
Yes
Check to see if
cellularity is
adequate. If not, use
more of the pellet, if
available and
prepare new slide.
No, continue to
next slide
Hologic Proprietary © 2013
Troubleshooting
Bloody or Proteinaceous Specimens
Does the slide have a
“halo” of cellular
material and/or white
blood cells?
Yes
No, continue to next
slide
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.
Hologic Proprietary © 2013
Troubleshooting
Bloody or Proteinaceous Specimens
Is the slide sparse and does
it contain blood, protein or
Yes-blood
non-cellular debris?
or noncellular
debris
No
Contact Hologic®
Technical Service
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.
Centrifuge remaining specimen from
PreservCyt vial, pour off. Add 30 ml of
Yes-protein 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.
Hologic Proprietary © 2013
Troubleshooting
Common Artifacts
•
•
•
•
Smudged Nuclear Detail
Compression Artifact
Staining Artifact
Edge of the Cylinder Artifact
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
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
Hologic Proprietary © 2013
Anatomy
Hologic Proprietary © 2013
Histology of the Epithelium
• The Thyroid is lined by
one type of epithelium:
• A single layer of
thyroid epithelial cells
called follicular cells
that are arranged in
spherical follicles
arranged around a
central ball of colloid
Courtesy of Prof. I. Salmon at Forpath vzw/asbl, oct; 1999
Hologic Proprietary © 2013
Specimen Adequacy
• Both the quantity and quality of the cellular
component as well as colloid must be
considered
• Guidelines for specimen adequacy vary. The
following is commonly used. A minimum of
six groups of well-visualized follicular cells,
with at least ten cells per group
– Note the following exceptions (Specimens with abundant
thick colloid and solid nodules containing cytologic atypia or
consisting only of abundant inflammatory cells should be
considered to be benign and satisfactory)
Hologic Proprietary © 2013
Normal Components and
Findings
• Follicular Cells
– Range in shape from cuboidal to columnar
– Nuclei are round to oval and are about the
size of a lymphocyte
– Evenly distributed granular chromatin
– Single, in honeycomb sheets and intact
follicles with even spacing
– Cytoplasm is fine and pale, stains blue with
Pap stain and blue/purple with Romanowsky
stain
Hologic Proprietary © 2013
Normal Components and
Findings
• Hürthle Cells
– Polygonal in shape and frequently binucleate
– Eccentrically placed nuclei ranging from
small to large
– Finely granular, abundant cytoplasm
staining blue to orange in Pap stain and
purple in Romanowsky stain
Hologic Proprietary © 2013
Normal Components and
Findings
• Flame Cells
– Cytoplasm is abundant and
vacuolated-with Romanowsky stain
these vacuoles show red to pink
staining material which is darker at
the edges
Hologic Proprietary © 2013
Normal Components and
Findings
• Multinucleated giant cells
– Commonly found with papillary carcinoma but not limited to only
this lesion
– Can be found in other benign and malignant conditions
• Lymphocytes
– May be present in both benign and malignant lesions
– Can be confused with stripped nuclei of follicular cells
– Will have coarser chromatin, a thin rim of cytoplasm, a less
prominent nuclear membrane and the presence of
lymphoglandular bodies
• Spindle Cells and Squamous Cells
– May be present in both benign and malignant lesions
Hologic Proprietary © 2013
Normal Components and
Findings
• Other findings
– Hemosiderin
• Associated with bleeding
• Present in aspirations of cyst and can help to favor a benign
thyroid nodule over a follicular neoplasm
• Stains golden brown with Pap stain and dark blue in
Romanowsky stain
– Calcification
• Dystrophic – Can be outline-like or coarse dense nodular
calcifications and can be found in both cysts as well as
follicular neoplasm
• Psammomatous – Concentrically laminated crystalline
structures associated with Papillary Carcinoma
Hologic Proprietary © 2013
Normal Components and
Findings
• Other Findings
– Mucin
• When present it’s thought that the aspirated lesion
is likely not located in the thyroid, however it may
also be associated with all types of thyroid cancers
– Amyloid
• Looks similar to dense colloid with a waxy
appearance
• Can be distinguished with Congo red stain
• Is associated with medullary carcinoma but can
also be present in amyloid goiter
Hologic Proprietary © 2013
Normal Components and
Findings
•
Colloid
– A honey like, gelatinous secretion that acts as a storage site for
thyroglobulin
– An active thyroid will produce a paler, thinner colloid. When less active,
the colloid with tend to be thicker and denser
– Stains blue, green, pink or orange with Pap stain and dark blue/purple
with Romanowsky stains
• Dense/solid
– Irregularly shaped rounded droplets
– Chicken wire appearance
– Often shows “stained glass cracking”
• Watery/diffuse
–
–
–
–
Thin membrane/cellophane /tissue paper like appearance
Cracked /mosaic pattern
Must be distinguished from serum
Can be lost on liquid-based cytology
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Copyright © 20123Hologic, All rights reserved.
Hologic Proprietary © 2013
Normal Components and
Findings
• Possible Contaminants
– Ciliated cells from thyroglossal duct cysts or by
accidental sampling of the trachea
– Transgressing blood vessels can be seen and are
sometimes present with Hürthle Cell Neoplasm
– Fat although rare may be present from the
subcutaneous adipose tissue in the neck and can
also rarely occur a range of thyroid lesions
– Skeletal Muscle will need to be distinguished from
dense colloid and will have striations and nuclei
Hologic Proprietary © 2013
Nondiagnostic Findings
• Cyst fluid only
• Obscuring factors
• Virtually acellular
– Exceptions - Specimens consisting primarily
of abundant thick colloid and solid nodules
containing cytologic atypia or consisting only
of numerous inflammatory cells should be
considered to be benign and satisfactory
Hologic Proprietary © 2013
Nondiagnostic Findings
• Cyst
– 15-25% of all thyroid nodules are cystic
– Exclusion of malignancy is not possible
with a diagnosis of cyst
– Benign and malignant thyroid lesions can
be cystic, Papillary carcinoma being the
most common cystic thyroid cancer
– Need to be wary of both false negative
and false positive diagnosis
Hologic Proprietary © 2013
Nondiagnostic Findings
• Cyst
– Abundant hemosiderin-laden macrophages
– Foamy histiocytes
– Blood
– Proteinaceous debris
– Watery colloid-amount will vary and be
difficult to appreciate
– Rare follicular cells may be present
• May show reactive/degenerative changes that
can mimic cancer
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Acute Thyroiditis
• Granulomatous Thyroiditis
– (Subacute, de Quervain’s)
– Fungal
• Aspergillus, Blastomyces, Candida, Pneumocystis
– Parasitic
• Echinococcus, Wucheria, Treponema
– Mycobacterial Thyroiditis
•
Tuberculosis
• Chronic Thyroiditis
– Lymphocytic Thyroiditis (Hashimoto)
– Riedel Thyroiditis/Disease
Hologic Proprietary © 2013
Benign Findings
• Acute Thyroiditis
– Very painful, potentially life threatening condition
– Very rare due to the accumulation of iodine in the thyroid
that acts as a “germ killer”
– Those at risk are the young, old, immunosuppressed and
malnourished
– Most common cause is bacterial and less likely fungal
– Staphylococcus aureus, Streptococcus pyogenes and
Streptococcus pneumoniae are responsible for
approximately 80% of cases
– Typically not aspirated, however if performed, aspiration
is a characteristic yellow-green pus
Hologic Proprietary © 2013
Benign Findings
• Acute Thyroiditis
– Abundant neutrophils and histiocytes
– Granulations tissue, necrosis and debris
– Scant epithelial cells, but when present
may have reactive/degenerative changes
– Bacteria may be noted
Hologic Proprietary © 2013
Benign Findings
• Granulomatous Thyroiditis (Subacute,
de Quervain’s)
– Commonly diagnosed clinically
– Most common cause of painful thyroid disease
and mainly affects middle aged women
– Possible viral etiology and may be a genetic
predisposition
– Primarily a self-limiting disease for most with
recovery in a few months using nonsteroidal
anti-inflammatory treatment
Hologic Proprietary © 2013
Benign Findings
• Granulomatous Thyroiditis (Subacute,
de Quervain’s)
– Typically hypocellular
– Tell tale multinucleated giant cells that are
engulfing colloid
– Loose clusters of epithelioid histiocytes
– Scant follicular cells which may show reactive
changes
– Background of lymphocytes, plasma cells,
eosinophils and neutrophils
Hologic Proprietary © 2013
Benign Findings
• Chronic Thyroiditis (Lymphocytic
Thyroiditis - Hashimoto)
• Most commonly seen form of thyroiditis
• Must be distinguished from MALT lymphoma
• Autoimmune disease most common in middle
aged women and adolescents
• Many patients don’t need to be aspirated and
can be diagnosed clinically, however
approximately one third of patients have atypical
presentation and will be biopsied
Hologic Proprietary © 2013
Benign Findings
• Chronic Thyroiditis (Lymphocytic
Thyroiditis - Hashimoto)
• Hypercellular aspirates
• Polymorphic lymphoid cells consisting of small
mature lymphocytes, larger reactive lymphoid
cells and occasional plasma cells
• Hürthle cells arranged in single cells and
sheets
Note: There is no minimum requirement for follicular or Hürthle
cell component to be considered satisfactory
Hologic Proprietary © 2013
Copyright © 2013Hologic, All rights reserved.
Hologic Proprietary © 2013
Copyright © 2013Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Chronic Thyroiditis (Riedel
Thyroiditis)
– Unknown origin affecting primarily middle
aged to older women, many of which have a
history of Hashimoto thyroiditis
– Most rare of all types of thyroiditis
– Epstein-barr virus could be a causative
agent
– Patient presents with a painless, non-tender
thyroid
Hologic Proprietary © 2013
Benign Findings
• Chronic Thyroiditis (Riedel
Thyroiditis)
– Frequently hypocellular to acellular
– Spindle mesenchymal cells
– Collagen strands
– May be a few lymphocytes, plasma cells,
neutrophils, eosinophils and rare giant
cells
Hologic Proprietary © 2013
Benign Findings
• Black Thyroid
– Associated with the antibiotic in the
tetracycline family given for acne
treatment called minocycline
– Stains similarly to that of melanin, but is
a break down product of the
minocycline
– Coarse dark brown/black pigment in the
cytoplasm of macrophages, follicular
cells and colloid
Hologic Proprietary © 2013
Benign Findings
• Benign Follicular Nodule(BFN) Overview
– Variable amounts of colloid
– Benign appearing follicular cells
– Hürthle cells
– Macrophages
Note: There may be rare microfollicles
present, but should be out numbered by
macrofollicles
Hologic Proprietary © 2013
Benign Findings
• Benign Follicular Nodule
– Nodular goiter
– Colloid nodule
– Hyperplasic (adenomatoid) nodule
– Follicular adenomas (macrofollicular
type)
– Grave’s disease
Hologic Proprietary © 2013
Benign Findings
• While this group of benign lesions have
different histological presentations, it is
difficult to distinguish them with FNA
• A diagnosis of BFN will warrant the same
conservative treatment regardless of which
specific histologic type of nodule
• A further sub-classification of type of
nodule may be used when possible in
addition to the diagnosis of BFN
Hologic Proprietary © 2013
Benign Findings
• Nodular Goiter
– Most commonly seen lesion in thyroid
FNA
– Can be distinguished from a follicular
neoplasm by the lack of microfollicles
and instead the follicular cells will be
arranged in sheets and macrofollicles
Hologic Proprietary © 2013
Benign Findings
• Nodular Goiter
–
–
–
–
Scant to moderate cellularity
Abundant colloid
Pigment laden or foamy histiocytes
Follicular cells are arranged in large flat honeycomb
sheets and in macrofollicles
– Nuclei of the follicular cells will be spaced uniformly
with in the sheet, centrally placed with in the
cytoplasm, small, round and bland appearing
– Naked nuclei may be in the background with in colloid
– Scattered Hürthle cells
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Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Colloid Nodule
– Type of nodular goiter that occurs
when follicular cells respond to the
extra release of TSH by the pituitary
gland by releasing and storing excess
colloid
– Little risk of malignancy
– However, a small malignant nodule
could be present next to the sampled
colloid nodule
Hologic Proprietary © 2013
Benign Findings
• Colloid Nodule
– Predominantly or consisting only of
abundant colloid
– Minimal cellularity - very few or no
follicular cells present
Note: Be sure to distinguish colloid
from serum
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Hyperplastic (adenomatoid)
Nodule
– Type of nodular goiter that occurs
when follicular cells respond to the
extra release of TSH by the pituitary
gland by excessive growth
Hologic Proprietary © 2013
Benign Findings
• Hyperplastic (adenomatoid)
Nodule
– Moderate cellularity
– Scant dense and watery colloid
(there may be a loss of watery colloid on
LBC and when present may appear tissue
paper like)
– Macrofollicles arranged in
honeycomb pattern
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Copyright © 2013 Hologic, All rights reserved.
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Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Follicular Adenoma (macrofollicular
type)
– Most common thyroid neoplasm
– Almost always occurs as a solitary nodule
while the remaining gland is normal
Hologic Proprietary © 2013
Benign Findings
• Follicular Adenoma (macrofollicular type)
– Can have variable cellularity
– Follicular cells will display benign features
however the nuclei can be enlarged, coarse
and hyperchromatic. The cytoplasm will be
finely granular and vacuolated
– Hürthle cells can be present
– Flame cell changes may be observed
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Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Benign Findings
• Grave’s Disease
•
•
•
•
Autoimmune disorder
Mainly affects middle aged women
Commonly diagnosed clinically due to hyperthyroidism
Diffuse rather than nodular enlargement in the majority
of patients
• FNA is not frequently performed
• Considered to be a risk factor for aggressive thyroid
cancer particularly when the nodule is cold with
papillary carcinoma being the most common
• The drugs given to treat this disease may cause
changes that can be confused with malignancy
Hologic Proprietary © 2013
Benign Findings
• Grave’s Disease
• Cellular aspirate
• Abundant pale watery colloid
• Follicular cells
–
–
–
–
Variable number
Large flat sheets and rarely microfollicles
Abundant, foamy cytoplasm
Enlarged vesicular nuclei with frequent with anisonucleosis and
prominent nucleoli
– Infrequently chromatin clearing and intranuclear grooves can be
seen which can be confused with papillary carcinoma
• If the patient has been treated, atypical features of the follicular
cells can be significant and confused with malignancy
• Lymphocytes (usually T cells) and oncocytes
• Flame cells
Hologic Proprietary © 2013
Atypical Findings
• Atypia of Undetermined Significance or
Follicular Lesion of Undetermined
Significance
• The degree of atypia associated with this category
is not severe enough to warrant a suspicious
diagnosis and is felt to be caused by a benign
process and/or the cellularity while adequate for
diagnosis, may be less than desirable
• Clinical correlation and repeat FNA is the
recommended follow up treatment
Hologic Proprietary © 2013
Atypical Findings
• Atypia of Undetermined Significance or Follicular Lesion of
Undetermined Significance-possible uses
• Predominance of microfollicles or Hürthle cells in a sparsely cellular
sample
• Abundant Hürthle cells in either a patient with known Hashimoto
thyroiditis or in a patient with multinodular goiter
• Air drying that causes poor nuclear detail, cyst lining cells that are
less elongated and more tightly packed and rare nuclear
enlargement with prominent nucleoli, all can mimic papillary
carcinoma
• Artificial crowding of follicular cells due to abundant blood and
clotting may mimic a follicular neoplasm
• Rare cells with mild atypia sometimes present in Hashimoto
thyroiditis
• An atypical lymphoid infiltrate (flow cytometry needed)
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Copyright © 2013 Hologic, All rights reserved.
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Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Suspicious Findings
• Suspicious for a Follicular Neoplasm
• Follicular carcinoma is the second most common malignancy
in the thyroid
• If well differentiated will have a good prognosis
• Difficult to distinguish between a benign follicular lesion and a
carcinoma on FNA
• Many FNA’s given this diagnosis will ultimately turn out to be
a benign proliferation
• FNA has shown to have a high sensitivity but low specificity
for follicular carcinoma and therefore considered a screening
test rather than a diagnostic test for this diagnosis
• Lobectomy or hemithyroidectomy is the follow up treatment
with this category where further histologic classification can
be made
Hologic Proprietary © 2013
Suspicious Findings
• Suspicious for a Follicular Neoplasm
•
•
•
•
•
•
•
•
Moderate cellularity with architectural alteration
Cell crowding
Overlapping
Follicular cells with round nuclei are arranged in
microfollicles, single cells and occasional trabeculae
Hyperchromasia, anisonucleosis and prominent nucleoli may
be present
Chromatin may be less granular than benign follicular cells
display and have an open appearance
Little to no colloid
Foamy and hemosiderin laden macrophages may be present
if cystic
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Copyright © 2013 Hologic, All rights reserved.
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Copyright © 2013 Hologic, All rights reserved.
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Copyright © 2013 Hologic, All rights reserved.
Hologic Proprietary © 2013
Malignant Findings
• Follicular Neoplasm, Hürthle Cell Type
– Uncommon subset of follicular neoplasm
composed predominantly of oncocytic
cells
– The majority of FNA’s diagnosed as
Hürthle cell neoplasm will be adenomas
rather than carcinomas
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Malignant Findings
• Follicular Neoplasm, Hürthle Cell Type
– Polygonal shaped cells arranged in a single cell pattern,
loosely cohesive or crowded three dimensional groups
– Abundant, dense, granular cytoplasm
– Nuclei are round and eccentrically placed with a
prominent central nucleoli
– Plasmacytoid appearance
– Occasional binucleation is seen
– Variation in both size of cells and size of nuclei can occur
– Colloid is either scant or absent
– Chronic inflammatory cells are absent
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Malignant Findings
• Papillary Carcinoma
•
•
•
•
•
Most Common malignancy in the thyroid
Can occur in any age range
More common in women that men
Good prognosis
Flat sheets can mimic benign cells. Nuclear
detail must be examined
• Many of the following criteria must be present in
order to make the diagnosis of papillary
carcinoma
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Malignant Findings
• Papillary Carcinoma
• Syncytial flat sheets and papillary groups
– Sometimes in a swirling like pattern
• Increased cellularity with crowding and overlapping
• Nuclei are enlarged, pale, round to oval or irregular in shape
and can display grooves and molding
• Chromatin is pale and powdery with micronucleoli
• Intranuclear cytoplasmic inclusions (can also be seen in other
thyroid neoplasms)
• Psammoma bodies are rare, but can be seen
• Multinucleated giant cells are often present
• Colloid may be present
• Hürthle cells and hemosiderin laden macrophages can be seen
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Malignant Findings
• Papillary Carcinoma Variants
– Variants have the same abnormal
features of papillary carcinoma, but show
some architectural, background or
cytoplasmic differences
– Some variants may have a different
prognosis for the patient
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Malignant Findings
• Papillary Carcinoma Variants
– Follicular
• Specimen consists of subtle features of papillary
carcinoma arranged in small to medium
microfollicles
– Cystic
• Follicular cells with the features of papillary
carcinoma arranged in small groups, sheets, follicles
or papillary arrangements that have abundant,
granular or vacuolated cytoplasm in a cystic
background of watery colloid and hemosiderin laden
macrophages
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Malignant Findings
• Papillary Carcinoma Variants
– Warthin-like
• Neoplastic cells are arranged in papillary groups,
have oncocytic cytoplasm and a background of
lymphocytes. When these lymphocytes are admixed
with neoplastic cells, be sure the changes represent
true malignant features and are not just reactive
changes.
– Oncocytic
• Abundant oncocytic/granular cytoplasm is the
dominating type of cytoplasm present in the
neoplastic cells with an absence of lymphocytes
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Malignant Findings
• Papillary Carcinoma Variants
– Tall Cell- (Aggressive type)
• At least half of the neoplastic cells are arranged in
papillary groups consisting of cells with the features of
papillary carcinoma, but are elongated (at least 2-3 times
long as wide) with abundant granular cytoplasm with
intranuclear cytoplasmic inclusions being more common
and often multiple. Occasional lymphocytes may be
seen
– Macrofollicular
• Subtle nuclear features of papillary carcinoma are found
in macrofollicular sheets that make up at least half of the
specimen
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Malignant Findings
• Papillary Carcinoma Variants
– Columnar Cell (Rare aggressive type)
• Cellular aspirate with elongated, columnar,
stratified, crowded cells that are typically
arranged in papillary groups, but can be seen in
flat sheets or clusters
• Longer in height than the tall cell variant
• Nuclei are hyperchromatic, oval and uniform with
discreet nucleoli
• Traditional features of papillary carcinoma are not
present
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Malignant Findings
• Papillary Carcinoma Variants
– Hyalinizing Trabecular
• Difficult to diagnose cytologically
• Tight groups of cells with a core of hyaline
stromal material
• Many of the same features of papillary
carcinoma including; psammoma bodies,
intranuclear cytoplasmic inclusions as well as
nuclear grooves
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Malignant Findings
• Medullary Thyroid Carcinoma
• Associated with other neuroendocrine tumors and although less
common than sporadic occurrences, can be hereditary
• A mutation in the RET proto-oncogene located at 10q11.2 is
responsible for the hereditary form of this malignancy
• Unlike other malignancies in the thyroid, medullary carcinoma
arises in the parafollicular C cells
• A combination of a follicular nodule with increased serum calcitonin
levels are findings that are indicative of medullary carcinoma
• Congo red stain for amyloid and immunohistochemical stains can
aid in the diagnosis with FNA
– Immunohistochemical staining;
» Calcitonin, CEA, chromogranin and synaptophysin positive (however
occasionally negative for calcitonin)
» Thyroglobulin negative (however occasionally positive)
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Malignant Findings
• Medullary Thyroid Carcinoma
• While not necessary to include subtype
in the diagnosis, there are many possible
variants which can be difficult to
distinguish from other malignancies;
– Small cell, papillary, follicular/glandular,
spindle cell, oncocytic, clear cell, giant cell,
mixed follicular/medullary(parafollicular),
neuroblastoma like, paraganglioma like,
angiosarcoma like, melanin producing,
amphicrine, squamous cell
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Malignant Findings
• Medullary Thyroid Carcinoma
• Single, dyshesive cells is the predominant pattern
although clusters, papillae and follicles can be
present
• Cells may be many different shapes including
round, polygonal, plasmacytoid and spindled
• Amyloid is frequently present and stains red with
Congo red stain, but when polarized light is applied
changes to apple-green
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Malignant Findings
• Medullary Thyroid Carcinoma
• Nuclei are commonly binucleated or multinucleated,
eccentrically located and typically round to oval with the
exception of the spindle cell variant in which case they will be
elongated
• Chromatin is coarsely granular with small nucleoli, but less
frequently can be prominent
• Cytoplasm can be variable, but is commonly abundant and
finely granular
• Nuclear grooves and nuclear pseudoinclusions may been seen
• Cytoplasmic granules stain red with Romanowsky stain, are
present in a large majority of cases, however when present are
only in a small portion of cells
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Malignant Findings
• Poorly Differentiated Carcinoma
• Rare, aggressive malignancy of follicular cell origin with
subtypes that can coexist including:
• Insular
• Non-insular
– Can arise as a transformation from a well differentiated
carcinoma (papillary or follicular)and then go onto further
transform into undifferentiated carcinoma (anaplastic)
– Patients usually present with advanced disease
– Lymph node and lung or bone metastasis is common
– Difficult to diagnose using FNA and are often called suspicious
for a follicular neoplasm
– Immunohistochemical staining;
• Thyroglobulin, TTF-1 and cytokeratin positive
• Calcitonin negative
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Malignant Findings
• Poorly Differentiated Carcinoma
– Aspirates are highly cellular
– Cells are arranged singly, in crowded groups, papillarylike aggregates or microfollicles, and can be wrapped by
endothelium, creating insulae or trabeculae
– Naked stripped nuclei, necrosis and mitoses are often
present
– Cytoplasm is scant and the cells often have a
plasmacytoid appearance
– Nuclei are round and frequently display variation in both
size and shape with irregular nuclear borders, granular
to coarse chromatin and can have varying sizes of
nucleoli
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Malignant Findings
• Undifferentiated Carcinoma (Anaplastic)
• Rapidly growing, rare, extremely aggressive tumor
• More common in women over age 50 than men
• Rapid tumor growth infiltrates into surrounding soft
tissues of the neck as well as cartilage and bone.
• Common site for metastasis is the lung
• A secondary co-existing thyroid carcinoma may
also be present in some cases
• Immunocytochemistry staining results are variable;
– Pan-keratin and vimentin often positive
– TTF-1 and thyroglobulin often negative
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Malignant Findings
• Undifferentiated Carcinoma (Anaplastic)
• Cellularity can vary and be decreased due to dense
fibrotic stroma and necrosis
• Cells are commonly arranged in single cells,
crowded groups and stripped nuclei
• Cells are round to polygonal or sometimes spindle in
shape with marked variation in size (can be giant)
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Malignant Findings
• Undifferentiated Carcinoma (Anaplastic)
• Nuclei are enlarged, hyperchromatic, with coarse,
irregular chromatin, irregular nuclear borders,
macronucleoli and intranuclear cytoplasmic
inclusions
• Abundant inflammatory cells, primarily neutrophils
can be present sometimes invading the cytoplasm
of tumor cells
• Frequent normal and abnormal mitoses
• Squamous differentiation may be present and
should be distinguished from Squamous cell
carcinoma
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Malignant Findings
• Metastatic Carcinomas and Lymphomas
• Metastases are uncommon, however when present, here are some of the
most common;
– Kidney- Must be distinguished from a follicular and Hürthle cell neoplasms
– Colorectal- Use classic criteria such as columnar shape to diagnose
– Lung- Papillary lesions may be confused with papillary thyroid carcinoma and
small cell ca may mimic insular carcinoma
– Breast- Must be distinguished from a follicular neoplasm
– Melanoma- Must be distinguished from medullary or anaplastic carcinoma
– Lymphoma- May be primary or secondary, the latter being more common. Can
be confused with undifferentiated carcinoma or Hashimoto thyroiditis
– Squamous cell carcinoma- May be primary or metastatic. Uncommon with a
poor prognosis primarily occurring in the elderly. Can be difficult to distinguish
from an undifferentiated carcinoma with squamous differentiation
• Patient history, flow cytometry (when considering a lymphoma) and
immunocytochemistry (TTF-1 and thyroglobulin) are invaluable in
diagnosing these cases
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Trademark Statement
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are registered trademarks of Hologic, Inc. and/or its
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All other trademarks are the property of their
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For more information…
• Visit our websites: www.hologic.com,
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- Product Catalog
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Bibliography
ThinPrep® 2000 Processor Operator’s Manual
ThinPrep 5000 Processor Operator’s Manual
Ali, Syed Z., Cibas, Edmund J.: The Bethesda System
for Reporting Thyroid Cytopathology Definitions,
Criteria and Explanatory Notes, 2010:1-171
Layfield, Lester: Cytopathology of the Head and Neck
ASCP Theory and Practice of Cytopathology Series
7, 1997: 159-208
Clark, Douglas P., Faquin, William C.: Thyroid
Cytopathology Second Edition, 2010: 1-200
DeMay, Richard M: The Art & Science of
Cytopathology 2nd Edition Superficial Aspiration
Cytology (Volume 2 of 4), 2012: 839-963
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Bibliography
Images:
Salmon, I. Prof. Normal Thyroid. Photograph. Forpath
vzw/asbl, oct; 1999
[http://www.forpath.org/workshops/9910/images/th02.jpg]
28 December 2012
SmartDraw® (Standard Edition) [Software]. (2011).
Retrieved from http://www.smartdraw.com
ThinPrep® Non-Gyn Morphology Reference Atlas:
Thyroid: 223-244
http://www.cytologystuff.com/learn/section2660.html
http://www.cytologystuff.com/learn/section2241.html
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