Last updated: 11/26/2023 Prepared by Dr.

Kurt Schaberg

Thyroid Cytology
Adequacy Criteria Must see at least 6 groups of well-visualized
follicular epithelial cells, each consisting of at
least 10 cells. (Ideally on one slide, but not req.)
Exceptions:
1) Abundant colloid with radiographic findings
compatible with a colloid nodule
2) Abundant inflammation with a solid nodule
(lymphocytes, granulomas, or neutrophils)
3) Atypia
Diff-Quick Normal cytology:
Follicular epithelium should be in nice big, flat
monolayered, macrofollicular sheets, with evenly
spaced (“Honeycomb-like”) cells. Minimal overlapping.
Sometimes see intact big 3-D entire spherical follicles.
Cells should have round nuclei with uniformly granular,
dark chromatin (think: “soccer ball”).
Nuclei are slightly bigger than an RBC.
If abundant granular cytoplasm ± nucleoli→ Hürthle
(Oncocytic) cells (a common change/metaplasia)
Pap
Colloid can appear “watery” as evenly spread
proteinaceous fluid, in “puddles” or “dense/thick” as 3D
Intact macrofollicle
hyaline globules.

Hints to thin, watery colloid include: Cracks (like glass),

Wrinkles (like cellophane), and rouleaux formation.
On ThinPrep: Looks like “tissue paper”

Watery Colloid

Dense/Thick Colloid
 Nondiagnostic
Used for specimens that don’t meet adequacy
criteria on first page.

Common scenarios:
Blood only (paucicellular)
Cyst fluid only
Proteinaceous fluid, debris, and hemosiderin-
laden histiocytes
Obscuring ultrasound gel
Bright purple globules (on diff quick)
Obscuring blood
Poor slide preparation/staining

Benign Lesions
Benign Follicular Nodular Disease “FND”
Histologically represent nodular goiter, adenomatoid nodules,
hyperplasia, and colloid nodules. Now, refer to as “Follicular
nodular disease” (FND) as may or may not be neoplastic.

Variable amounts of: colloid (the more the better! ;-), bland
follicular cells, Hürthle cells, and macrophages.

Should be sparse to moderately cellular with a good amount of

colloid (often easiest to see on diff-quick).
Occasional microfollicles are permissible.
A little anisonucleosis is ok (attributable to endocrine atypia) as long
as there is no nuclear contour irregularities or chromatin clearing

Frequently see Cystic degeneration: Changes include macrophages,

“reparative” stretched cyst lining epithelial cells. These cells are
elongated, enlarged, and have finely granular chromatin and
squamoid or spindled shape/cytoplasm. Low N:C ratios. Can mimic
malignancy/AUS, but can forgive if focal and mild.

Classic, reassuringly benign

findings: A ton of colloid, with
scattered bland follicular
epithelium ☺

Cyst lining cells

Lymphocytic Thyroiditis
Generalized term that encompasses many conditions, the
most common of which is chronic lymphocytic
(Hashimoto’s) thyroiditis. Often middle-aged women with
associated circulating autoantibodies.
Hypercellular smear.
Abundant, polymorphic lymphocytes.
Hürthle/Oncocytic metaplasia common
(Large cells with abundant granular cytoplasm and
prominent nucleoli).
Advanced cases may be hypocellular (due to fibrosis).
May have significant anisonucleosis.
No minimum quantity of follicular cells required for
adequacy.

Granulomatous Thyroiditis aka subacute or de Quervain’s

Self-limited inflammatory condition, usually diagnosed
clinically.

Clusters of epithelioid histiocytes (i.e., granulomas)

and multinucleated giant cells, often ingesting colloid.
Early can have neutrophils and eosinophils.
Later stages are hypocellular and have lymphocytes.

Graves Disease
Autoimmune thyroid hyperplasia/hyperthyroidism.
Although usually diffuse, can have nodules.

Non-specific findings, on spectrum with FND.

Abundant, delicate, foamy cytoplasm.
Enlarged nuclei with prominent nucleoli.
“Flame cells” have marginal vacuoles with red/pink
frayed edges.
Other Benign Disorders
Pigment: Can see lipofuscin and/or hemosiderin commonly. Can see “black thyroid” pigmentation with
tetracycline family antibiotics.
Amyloid goiter: Diffuse amyloid deposition in thyroid gland. Can be 1° or 2°. Amyloid has amorphous
look of colloid, but has embedded fibroblasts.
Acute Suppurative Thyroiditis: Bacterial infection→ present with fever and neck pain. Smears show
fibrinopurulent debris (abscess).
Riedel Thyroiditis: IgG4-related disease of the thyroid with progressive fibrosis. Hypocellular smears.
Tumors
Papillary Thyroid Carcinoma “PTC”
Most common malignant thyroid neoplasm
Relatively good prognosis. Spreads via lymphatics to
lymph nodes.

Architecture:
-Papillary structures w/ and w/o fibrovascular cores
-May be in monolayered sheets or 3D groups
-Can see cellular swirls (“cartwheel” pattern)
Papillary architecture
Nuclear features:
-Enlarged and crowded nuclei, often molded
-Nuclear overlapping, elongation
-Irregularly shaped to oval nuclei
-Longitudinal nuclear grooves
-Intranuclear (cytoplasmic) pseudoinclusions (INPI)
-Powdery, pale chromatin
-Marginal micronucleoli
-Thick nuclear membranes

Other features:
-Dense, squamoid cytoplasm Pseudoinclusions
-Multinucleated giant cells
-Dense, “Bubble gum” colloid
-Septate cytoplasmic vacuoles
-Psammoma bodies
-Oncocytic metaplasia
-“Histiocytoid” and “hobnail” cells

Nuclear crowding/overlapping

Bubble gum colloid

Irregular contours
 Follicular Neoplasm “FN”
Cannot differentiate between Follicular Adenoma and
Carcinoma on cytology specimens (need to see capsular
or vascular invasion on resection specimen!), so one
overarching cytology Dx is given.

Moderately or Markedly cellular

Significant alteration in follicular architecture (i.e. non-
macrofollicular)
→ Repetitive microfollicular pattern, single cells, and/or
cell crowding/overlapping in trabeculae.
→ This should be the dominant pattern to Dx FN
(occasional microfollicles are acceptable in FND) Microfollicle: less than 15 cells
→ Minimal colloid arranged in a circle that is at least 2/3
Usually minimal cytologic atypia. complete

If less cellular→ consider AUS

If cytologically malignant→ consider “Suspicious for
malignancy” or just “Malignant”

Note: The Bethesda system no longer endorses saying

“Suspicious for FN” even though up to 1/3 of cases will be
non-neoplastic, hyperplastic nodules… just put in a
disclaimer for that possibility in your report I guess
¯\_(ツ)_/¯

Oncocytic (Hürthle cell) Follicular Neoplasm “FN-OFN”

Exclusively oncocytes (or almost exclusively)
→Abundant granular cytoplasm, enlarged round
nucleus, prominent nucleolus
Moderately or Markedly cellular
Significant alteration in follicular architecture (i.e. non-
macrofollicular)
→ Single cells, sheets and/or trabeculae.
Minimal colloid
Transgressing blood vessels
Absence of inflammation
Atypia/Anisonucleosis
→ Large oncocytes with ≥2x variability nuclear size
→ Small oncocytes with high N:C ratios
→ Binucleation is common
→ Careful though: While oncocytic atypia is pretty much
always seen in malignancy, atypical oncocytes can be seen in
FND and lymphocytic thyroiditis. However, if there is NOT
atypia, consider downgrading to AUS or benign (Not specific,
somewhat sensitive)
 Medullary Carcinoma
Rare. Can be sporadic or inherited (part of MEN 2A&B)
Derived from Parafollicular C cells→ stain with Calcitonin!
RET mutations (somatic or germline)

Moderate to Marked Cellularity. Often discohesive.

Plasmacytoid, polygonal, to spindled cells.
Mild to moderate pleomorphism.
“Salt and Pepper” chromatin. Can have intranuclear inclusions.
Granular cytoplasm with small granules.
Occasional amyloid fragments

IHC: (+) Calcitonin, Synaptophysin/Chromogranin, TTF1, ±PAX8

(-) Thyroglobulin

Poorly-Differentiated Thyroid Carcinoma

Thyroid carcinomas with necrosis and high mitotic activity that have an intermediate prognosis between
well-differentiated thyroid carcinomas (e.g., PTC) and Anaplastic carcinoma.
Uniform population of cells. High N:C ratios. Cellular smears. Scant/absent colloid.
Apoptoses, mitoses, and/or necrosis.
Should consider/exclude a metastasis.
Often hard to specifically diagnose on FNA. Often diagnosed as “malignant” or “follicular neoplasm,”
etc… Compared to Anaplastic carcinoma, which also has mitoses and necrosis, PDTC does not have
extreme pleomorphism or Sarcomatoid features.

Undifferentiated (Anaplastic) Thyroid Carcinoma

Extremely aggressive. Poor prognosis (<1yr survival).
Classically older women with rapidly growing, hard neck
mass → trouble breathing
Variable cellularity. Often discohesive.
Epithelioid to Spindled cells.
Enlarged, pleomorphic nuclei. Chromatin clumping.
Often associated necrosis and inflammation (often PMNs).
Can see osteoclast-like giant cells.
Abundant mitoses, often abnormal.
Squamous cell carcinoma is considered a subtype (but have
to exclude a metastasis!)
IHC: (+) Pancytokeratin, PAX8; (-) TTF1, Thyroglobulin
 Metastases/Other Tumors
Most common sources of metastases: Lung, Breast, Melanoma, Colon, Kidney.
Lymphoma: Most common are Diffuse Large B-Cell Lymphoma (DLBCL) and Extranodal Marginal Zone B-
Cell Lymphoma of Mucosa Associated Lymphoid Tissue (MALT). Usually in the setting of Chronic
lymphocytic thyroiditis.
Rare primary tumors (pretty much anything! ;-): Mucoepidermoid carcinoma, Thymoma, SETTLE, etc…

Parathyroid Adenoma
Often clinically elevated serum Calcium and PTH.
Often deep/posterior, but may be intrathyroidal.
No colloid. Trabecular/packeted fragments.
May have triangular “wedge” shapes.
Hypercellular with Small monotonous cells.
Round nuclei. Salt and pepper chromatin.
Dispersed individual cells in the background.

IHC: (+) GATA3, PTH, Chromogranin (less so, synaptophysin)

(-) TTF1, Thyroglobulin
Cyst fluid and/or a needle rinse can be sent for PTH levels

Regarding “NIFTP”
Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) cannot be reliably
separated from the Follicular variant of PTC (FVPTC) by cytology. NIFTP usually has microfollicular or
crowded architecture with mild nuclear changes.

To avoid overtreating NIFTP, which is indolent, follicular-patterned aspirates with nuclear changes that raise
the possibility of FVPTC or NIFTP (e.g., mild nuclear changes) are best classified as “Follicular neoplasm.”
Findings that take NIFTP out of consideration: 1) Papillary architecture, 2) Psammoma bodies, 3) frequent
pseudoinclusions, 4) Sheet-like architecture

Atypia of Undetermined Significance (“AUS”)

Indeterminate category for cases with one or more findings that raise concern for malignancy, but are
insufficient for a more definitive diagnosis. Only low risk of malignancy.
Usually either mild or focal nuclear changes or architectural changes.
Only fair interobserver reproducibility.
Usually treated with follow up with repeat FNA and/or molecular testing.
Possible scenarios:
Sparsely cellular aspirate with lots of microfollicles or Hürthle cells (A specimen needs to be markedly
cellular to Dx a FN)
Mild PTC nuclear changes (e.g., some enlargement, grooves, and/or powdery chromatin)
Psamommatous calcifications without other PTC findings
Atypical lymphocytes concerning for lymphoma
 Suspicious for Carcinoma
Indeterminate category used when the features raise a strong suspicion for malignancy, but the findings
are not conclusive for a definitive diagnosis. High risk of malignancy.
Mainly useful to maintain the high PPV of a “malignant” diagnosis.
Frequently treated with surgery (lobectomy vs total thyroidectomy).
Sometimes may employ molecular or repeat FNA, particularly when planning extent of surgery.

Molecular
For AUS or FN aspirates, molecular testing can be useful to risk stratify lesions to determine the risk of
malignancy and next steps in treatment.
Papillary Thyroid Carcinoma: MAPK Pathway: BRAF (most classic PTC’s), V600E (most common)
RAS (associated with follicular variant & NIFTP), ALK, NTRK fusions

Medullary Carcinoma: RET (think MEN2A&B)

Follicular Neoplasms (FA & FN): RAS point mutations most common, PPARG rearrangements (often with
PAX8), PTEN

Poorly Differentiated and Anaplastic: (Additional, late events, more aggressive tumors)
TP53, CTNNB1, TERT promoter mutations, ATK1, PIK3CA

The Bethesda System

Try to put all FNAs into one of these categories to help the clinicians determine the next appropriate
steps in management.

Diagnostic Category Risk of Management

Malignancy
I Unsatisfactory/ ~15% Repeat US-guided FNA
Nondiagnostic

II Benign ~5% Clinical/US follow-up

III AUS ~25% Repeat FNA and/or Molecular

testing

IV Follicular Neoplasm ~30% Lobectomy

V Suspicious for ~75% Thyroidectomy

Malignancy

VI Malignant ~97% Thyroidectomy