thinprep non-gyn lecture series - cytology stuff preparation troubleshooting • after staining, you...
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Title
Hologic Proprietary © 2013
ThinPrep® Non-Gyn Lecture Series
Thyroid Cytology
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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 • Minimizes number of slides per patient • Offers versatility to perform ancillary testing
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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
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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®
• Balanced electrolyte solutions such as -Plasma-Lyte® -Polysol®
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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 © 2013Hologic, All rights reserved.
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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 temp. • 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 prior
to processing • PreservCyt Solution cannot be substituted
with any other reagents • Cells in PreservCyt Solution are preserved for
up to 3 weeks at a temperature range of 4°-37°C
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FNA Biopsy
• Performed by a cytopathologist or clinician. • A 23 gauge or 25 gauge needle with a 10ml
syringe is used • The area is cleaned with 95% ethanol • The needle is passed thru the skin and into the
nodule. Several strokes are made with or without vacuum created by the syringe plunger
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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 Plasma-Lyte® 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.
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Sample Preparation
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® to cell pellet and repeat from step 2
5. Add recommended # of drops of specimen to PreservCyt® 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 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 on 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 cell pellet, 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® vial (# of drops added is dependent on sample volume; see future slides)
- If cell pellet is distinctly red or brown (indicating presence of blood), conduct a CytoLyt® wash
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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 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 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 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 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.
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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.
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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.
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Troubleshooting Common Artifacts
• Smudged nuclear detail • Compression artifact • Staining artifact • Edge of the cylinder artifact
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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
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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
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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, fresh alcohol baths or an
additional rinse step after the cytoplasmic stains is required
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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
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Anatomy
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Histology of the Epithelium
• The Thyroid is lined by one type of epithelium: • A single layer of
thyroid epithelial cells called follicular cells are arranged in spherical follicles around a central ball of colloid
Courtesy of Prof. I. Salmon at Forpath vzw/asbl, oct; 1999
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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 : A minimum number of follicular cells are not required for
the following exceptions: Specimens consisting primarily of abundant thick colloid and solid nodules containing cytologic atypia or consisting of numerous inflammatory cells should be considered satisfactory for evaluation
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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
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Normal Components and Findings
• Hürthle Cells – Polygonal in shape and frequently bi-
nucleate – Eccentrically placed nuclei ranging from
small to large – Finely granular, abundant cytoplasm
staining blue to orange with Pap stain
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Normal Components and Findings
• Multinucleated giant cells – Commonly found with papillary carcinoma
but not limited to this lesion – Can be found in other benign and malignant
conditions
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Normal Components and Findings
• 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, less prominent nuclear membrane and the presence of lymphoglandular bodies
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Normal Components and Findings
• Spindle cells and squamous cells – May be present in both benign and
malignant lesions
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Normal Components and Findings
• Calcifications – Dystrophic
• Peripheral “eggshell” or rim-like – Usually found in benign cysts but can be found with
follicular neoplasms
• Coarse, dense nodular – Psammomatous
• Concentrically laminated, crystalline structures associated most often with papillary carcinoma
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• Hemosiderin – Associated with bleeding – Present in cyst aspirations and can help to
favor a benign thyroid nodule over a follicular neoplasm
– Stains golden-brown with Pap stain
Normal Components and Findings
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Normal Components and Findings
• Amyloid – Similar to dense colloid: waxy appearance – Can be distinguished with Congo red stain – Associated with medullary carcinoma, but
also present in amyloid goiter
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Normal Components and Findings
• Mucin – When present it’s thought that the
aspirated lesion is likely not located in the thyroid, however it may be associated with all types of thyroid cancers
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Normal Components and Findings
• Colloid – A glycoprotein that is the storage site for iodinated thyroid
hormones – An active thyroid produces a paler, thinner colloid – With less active thyroid, colloid tends to be thicker and
denser – Stains blue, green, pink, or orange with Pap stain
• Dense/solid
– Irregularly shaped, rounded droplets – Often shows perpendicular cracking
• Watery/diffuse
– Thin, membrane/cellophane/tissue paper-like
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Normal Components and Findings
• Possible Contaminants – Ciliated cells from thyroglossal duct cysts or
by accidental sampling of the trachea – Fat, although rare, may be present from the
subcutaneous adipose tissue in the neck and can also rarely occur in a range of thyroid lesions
– Skeletal Muscle needs to be distinguished from dense colloid; has striations and nuclei
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Nondiagnostic Findings Types
• Cyst fluid only • Obscuring factors • Virtually acellular
– NOTE : A minimum number of follicular cells are not required for the following exceptions: Specimens consisting primarily of abundant thick colloid and solid nodules containing cytologic atypia or consisting of numerous inflammatory cells should be considered to be satisfactory for evaluation
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Nondiagnostic Findings Overview
• 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
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Nondiagnostic Findings Cytology
• Cyst – Abundant hemosiderin-laden macrophages – Foamy histiocytes – Blood – Proteinaceous debris – Watery colloid - amount will vary and may be
difficult to appreciate – Rare follicular cells may be present
• May show reactive/degenerative changes that can mimic cancer
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• Acute thyroiditis • Granulomatous thyroiditis
– Subacute (de Quervain’s) – Fungal
• Aspergillus, Blastomyces, Candida, Pneumocystis – Parasitic
• Echinococcus, Wucheria, Treponema – Mycobacterial thyroiditis
• Tuberculosis
• Chronic Thyroiditis – Lymphocytic (Hashimoto) thyroiditis – Riedel thyroiditis/disease
Benign Findings-Inflammatory Types
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Benign Findings-Inflammatory Overview
• Acute Thyroiditis – Very painful, potentially life threatening – Very rare due to the accumulation of iodine
in the thyroid which acts as a “germ killer” – Those at risk are the young, old,
immunosuppressed, and malnourished
continued on next slide
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• Acute Thyroiditis - continued – Most common cause is bacterial (less
commonly fungal) – Staphylococcus aureus, Streptococcus
pyogenes, and Streptococcus pneumoniae are responsible for approximately 80% of cases
– Typically not aspirated. If performed, aspirate is a characteristic yellow-green pus
Benign Findings-Inflammatory Overview
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Benign Findings-Inflammatory Cytology
• Acute Thyroiditis – Abundant neutrophils and histiocytes – Granulation tissue, necrosis, and debris – Scant epithelial cells. When present may
have reactive/degenerative changes – Bacteria may be noted
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Benign Findings-Inflammatory Overview
• Granulomatous thyroiditis (Subacute, de Quervain’s) – Usually diagnosed clinically (without FNA) – Most common cause of painful thyroid disease – Mainly affects middle-aged women – Possible viral etiology; possible genetic predisposition – Self-limiting disease for most: recovery in a few
months – Symptomatic relief with nonsteroidal anti-inflammatory
agents
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Benign Findings-Inflammatory Cytology
• Granulomatous thyroiditis (Subacute, de Quervain’s) – Typically hypocellular – Telltale multinucleated giant cells engulfing colloid – Granulomas and loose clusters of epithelioid
histiocytes – Scant follicular cells which may show reactive
changes – Background of lymphocytes, plasma cells,
eosinophils, and neutrophils
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Benign Findings-Inflammatory Overview
• Chronic lymphocytic (Hashimoto) thyroiditis • Most common 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 • One-third of patients have atypical presentation and
are biopsied
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Benign Findings-Inflammatory Cytology
• Chronic lymphocytic (Hashimoto) thyroiditis • Hypercellular • Polymorphic lymphoid cells: small mature
lymphocytes, larger reactive lymphoid cells, occasional plasma cells
• Hürthle cells (isolated cells and in sheets) Note: There is no minimum requirement for follicular or Hürthle cell
component to be considered satisfactory
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• Riedel Thyroiditis – Unknown cause – Primarily middle-aged or older women, many of
whom have a history of Hashimoto thyroiditis – Most rare of all types of thyroiditis – Epstein-Barr virus could be a causative agent – Patients present with a painless, non-tender
thyroid
Benign Findings-Inflammatory Overview
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Benign Findings-Inflammatory Cytology
• Riedel Thyroiditis – Frequently hypocellular to acellular – Spindle-shaped mesenchymal cells – Collagen strands – May be a few lymphocytes, plasma cells,
neutrophils, eosinophils, and rare giant cells
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Benign Findings-Inflammatory Overview & Cytology
• Black Thyroid – Associated with minocycline, an antibiotic in
the tetracycline family given for acne treatment – Similar appearance to melanin, but is a
breakdown product of minocycline – Coarse, dark brown/black pigment in the
cytoplasm of macrophages, follicular cells, and colloid
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Benign Findings-Epithelial Types
• Benign Follicular Nodule (BFN) – Nodular goiter – Colloid nodule – Hyperplastic (adenomatoid) nodule – Follicular adenoma (macrofollicular
type) – Graves’ disease
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Benign Findings-Epithelial Overview
• Benign Follicular Nodule (BFN): – Although these benign lesions have different
clinical and histologic features, they are impossible to distinguish by FNA
– A diagnosis of BFN warrants the same conservative treatment regardless of the specific histologic classification
– Histologic terms like “c/w colloid nodule” or “c/w adenomatoid nodule” are acceptable modifiers under the Benign general categorization
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Benign Findings-Epithelial Cytology
• Benign Follicular Nodule (BFN): – Variable amounts of colloid – Benign-appearing follicular cells – Hürthle cells – Macrophages Note: There may be rare microfollicles, but they should
be out numbered by macrofollicles
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Benign Findings-Epithelial Overview
• Nodular Goiter (NG) – Most common thyroid nodular disease – Nodules are usually comprised mostly of
macrofollicles – A minority of nodules in NG are comprised
mostly of microfollicles; these are often classified as abnormal by FNA (“false positives”)
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Benign Findings-Epithelial Cytology
• Nodular Goiter – Scant to moderate cellularity – Abundant colloid – Pigment-laden and/or foamy histiocytes – Follicular cells arranged in variably sized
sheets (macrofollicular fragments) and in intact macrofollicles
continued on next slide
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Benign Findings-Epithelial Cytology
• Nodular Goiter - continued – Nuclei of the follicular cells uniformly
spaced within the sheet, centrally placed within the cytoplasm, small and round
– Naked nuclei – Scattered Hürthle cells
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Benign Findings-Epithelial Overview
• Colloid Nodule – Subtype of nodular goiter nodule that has
markedly enlarged follicles filled with abundant colloid
– Little risk of malignancy – However, a small (usually biologically
insignificant) malignant nodule could be present next to the sampled colloid nodule
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Benign Findings-Epithelial Cytology
• Colloid Nodule – Abundant colloid – Minimal cellularity: very few or no follicular
cells present
Note: Be sure to distinguish colloid from serum
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Benign Findings-Epithelial Overview
• Hyperplastic (adenomatoid) nodule – Subtype of nodular goiter nodule that consists
predominantly of microfollicles – Represents a minority of nodular goiter
nodules
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Benign Findings-Epithelial Cytology
• Hyperplastic (adenomatoid) nodule – Moderate to marked cellularity – Scant colloid – Predominance of microfollicles Note: Such cases are interpreted as “Follicular
Neoplasm/Suspicious for Follicular Neoplasm” and represent one of the major limitations of FNA.
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Benign Findings-Epithelial Overview
• Follicular Adenoma (macrofollicular type) – Most common thyroid neoplasm – Almost always a solitary nodule
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Benign Findings-Epithelial Cytology
• Follicular Adenoma (macrofollicular type) – Variable cellularity – Follicular cells display benign features – Nuclei can be enlarged, coarsely granular,
and hyperchromatic – The cytoplasm is finely granular – Hürthle cell variant
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• Graves’ disease • Autoimmune disorder • Mainly affects middle-aged women • Commonly diagnosed clinically due to
hyperthyroidism • Diffuse rather than nodular enlargement in
the majority of patients
continued on next slide
Benign Findings-Epithelial Overview
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• Graves’ disease - continued • FNA rarely performed • Risk factor for aggressive thyroid cancer,
particularly when the nodule is cold (Papillary carcinoma the most common)
• Drugs given to treat this disease may cause changes that can be confused with malignancy
Benign Findings-Epithelial Overview
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• Graves’ Disease • Cellular aspirate • Follicular cells
– Variable number – Large flat sheets and rarely microfollicles – Abundant, foamy cytoplasm – Enlarged vesicular nuclei with frequent anisonucleosis
and prominent nucleoli – Infrequently, chromatin clearing and intranuclear grooves
can be seen which can be confused with papillary carcinoma
continued on next slide
Benign Findings-Epithelial Cytology
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• Graves’ Disease - continued • Abundant pale watery colloid • If the patient has been treated, atypical
follicular cells can be present and confused with malignancy
• Lymphocytes (usually T cells) and Hürthle cells
• Flame cells (Romanowsky stains)
Benign Findings-Epithelial Cytology
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Atypia of Undetermined Significance Overview
• Alternative name: Follicular Lesion of Undetermined Significance • The degree of atypia is not severe enough
to warrant a suspicious or malignant diagnosis
• Clinical correlation and repeat FNA are the usual management
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Several different patterns: • Predominance of microfollicles in a
sparsely cellular sample • Exclusively Hürthle cells in a sparsely
cellular sample • Exclusively Hürthle cells in a patient with
Hashimoto thyroiditis or multinodular goiter
continued on next slide
Atypia of Undetermined Significance Cytology
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Several different patterns - continued • Atypia obscured by preparation artifact;
atypical cyst lining cells; marked nuclear enlargement with prominent nucleoli
• Cells with mild nuclear atypia (e.g., in Hashimoto thyroiditis)
• Atypical lymphoid infiltrate (flow cytometry needed)
Atypia of Undetermined Significance Cytology
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Suspicious Findings Types
• Suspicious for Follicular Neoplasm Synonym: Follicular Neoplasm
• Suspicious for Follicular Neoplasm, Hürthle cell type
Synonym: Follicular Neoplasm, Hürthle cell type • Suspicious for Papillary Carcinoma • Suspicious for Medullary Carcinoma
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• Follicular carcinoma is the second most
common malignancy of the thyroid • If well-differentiated, a good prognosis • Impossible to diagnose invasion on FNA,
so can only triage worrisome patterns to surgery
continued on next slide
Suspicious for Follicular Neoplasm Overview
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continued • Most FNAs triaged to surgery with this
diagnosis prove to be benign • FNA has high sensitivity but low specificity
for follicular carcinoma, therefore it’s considered a screening rather than diagnostic test for this diagnosis
• Lobectomy is the usual management for this diagnosis
Suspicious for Follicular Neoplasm Overview
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Suspicious for Follicular Neoplasm Cytology
• Moderate to marked cellularity • Little or no colloid • Significant architectural changes:
–Predominance of microfollicles and/or trabeculae
• Cell crowding and overlapping • Hyperchromasia, anisonucleosis, and
prominent nucleoli may be present but are not essential for diagnosis
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Suspicious for Follicular Neoplasm, Hürthle Cell Type
Overview
• Uncommon subset of follicular neoplasm
composed exclusively (or virtually exclusively) of oncocytic cells
• The majority prove to be adenomas rather than carcinomas
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• Polygonal cells • Isolated cells or three-dimensional
groups (loosely cohesive or crowded) • Abundant dense, granular cytoplasm • Nuclei are round and eccentrically placed
with a prominent central nucleolus
continued on next slide
Suspicious for Follicular Neoplasm, Hürthle Cell Type
Cytology
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continued • Plasmacytoid appearance • Occasional binucleation • Variation in size of cells and nuclei can
occur • Colloid scant or absent • No increase in lymphocytes
Suspicious for Follicular Neoplasm, Hürthle Cell Type
Cytology
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Suspicious for Papillary Carcinoma Overview
• Suspicious for Papillary Carcinoma • Subjective diagnosis used when criteria
falls short of a malignant diagnosis • Suboptimal sampling • Suboptimal preservation • Unusual presentation • Insufficient quality or quantity
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First pattern: – A moderate to highly cellular specimen
composed of benign follicular cells that are arranged predominantly in macrofollicular sheets. Only some of the cells display the following features of papillary carcinoma; • Nuclear enlargement, pallor, grooves, membrane
irregularities and/or molding with the exception of intranuclear pseudoinclusions which are rare or absent
Suspicious for Papillary Carcinoma Cytology
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Second pattern: – A specimen with variable cellularity displaying
the following nuclear changes associated with papillary carcinoma, however; • Nuclear enlargement and pallor are only mild to
moderately displayed • Nuclear membrane irregularities, molding and
intranuclear pseudoinclusions are minimal or absent
• Nuclear grooves are conspicuous
Suspicious for Papillary Carcinoma Cytology
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Third pattern: – A very sparsely cellular sample with many
features of papillary carcinoma present
Suspicious for Papillary Carcinoma Cytology
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Fourth pattern: – Follicular cells are arranged in groups and
sheets – Nuclei are enlarged and pale with some grooves – Intranuclear pseudoinclusions are rare or
absent – Hemosiderin laden macrophages are present – Large, vacuolated atypical histiocytoid cells with
enlarged nuclei – Rare psammoma body like calcifications
continued on next slide
Suspicious for Papillary Carcinoma Cytology
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Suspicious for Medullary Carcinoma Overview & Cytology
• Subjective diagnosis used when criteria falls short of a malignant diagnosis
• Suboptimal preservation can lead to smudged chromatin and overall cell degeneration
• Can be confused with a lymphoid lesion due to the single cell population of loosely cohesive/single cells with high n/c ratios
• Insufficient quantity or unusual presentation • Immunocytochemical stains and serum
calcitonin level of the patient can be helpful
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Malignant Findings Types
• Papillary Carcinoma • Hyalinizing Trabecular Tumor • Medullary Thyroid Carcinoma • Poorly Differentiated Carcinoma • Undifferentiated (Anaplastic) Carcinoma • Squamous Cell Carcinoma • Primary Lymphomas • Metastatic Carcinomas and Lymphomas
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Malignant Findings Overview
• Papillary Carcinoma • Most common malignancy of the thyroid • Can occur at any age, including childhood • More common in women than men • Good prognosis • Flat sheets mimic benign macrofollicular
fragments. 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 Cytology
• Papillary Carcinoma • Syncytial-like flat sheets and papillary groups
– Sometimes in a swirling pattern • Increased cellularity with crowding and
overlapping • Nuclei are enlarged, pale, round to oval or
irregular in shape and display grooves and molding
• Chromatin is pale and powdery with micronucleoli continued on next slide
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Malignant Findings Cytology
• Papillary Carcinoma - continued • Intranuclear cytoplasmic inclusions (can also
be seen in other thyroid neoplasms) • Psammoma bodies are rare • Multinucleated giant cells often present • Colloid may be present • Hürthle cells and hemosiderin-laden
macrophages can be seen
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• Papillary Carcinoma Variants • Variants have the same abnormal features of
papillary carcinoma, but with architectural, background, or cytoplasmic differences
• Some variants carry a different prognosis • Not necessary to subtype papillary carcinoma by
FNA because the initial management of all subtypes is the same (total thyroidectomy)
• Histopathologic examination permits definitive subtyping
Malignant Findings Overview
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• Papillary Carcinoma Variants – Follicular variant
• Nuclear features are more subtle • Cells arranged in microfollicles
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Cystic variant
• Neoplastic follicular cells have abundant granular or vacuolated cytoplasm
• Cystic background of watery colloid and hemosiderin-laden macrophages
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Warthin-like variant
• Neoplastic cells have oncocytic cytoplasm • Increased number of lymphocytes. When lymphocytes are admixed with neoplastic cells, be sure
the changes are truly malignant and not just reactive
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Oncocytic variant
• Abundant oncocytic (granular) cytoplasm dominates
• No increase in lymphocytes
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Tall cell variant
• At least half of the neoplastic cells are elongated (at least 2-3 times as long as they are wide)
• Abundant granular cytoplasm • Intranuclear cytoplasmic inclusions are frequent
and often multiple. Occasional lymphocytes may be seen
• Worse prognosis
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Macrofollicular variant
• Nuclear features are more subtle • Macrofollicular sheets make up at least half of
the specimen
Malignant Findings Cytology
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• Papillary Carcinoma Variants – Columnar cell variant
• Elongated, columnar, stratified, crowded cells • Less cytoplasm than the tall cell variant (higher
n:c ratio) • Nuclei are hyperchromatic, oval, and uniform • Discreet nucleoli • Nuclear features of papillary carcinoma are more
subtle • Very rare • Worse prognosis
Malignant Findings Cytology
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• Hyalinizing Trabecular Tumor • Controversial, rare neoplasm • Some consider it a variant of papillary
carcinoma • Difficult to diagnose cytologically • Histopathology: tight groups of cells
with a core of hyaline stromal material
continued on next slide
Malignant Findings Overview & Cytology
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• Hyalinizing Trabecular Tumor-continued
• Many of the same features of papillary carcinoma including: psammoma bodies, intranuclear cytoplasmic inclusions, nuclear grooves
• Most are interpreted as malignant or suspicious for papillary carcinoma by FNA
Malignant Findings Overview & Cytology
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Malignant Findings Types
• Medullary Thyroid Carcinoma • Many histologic variants (not necessary to subtype)
– 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 Overview
• Medullary Thyroid Carcinoma • Sporadic or familial • Tumor of the parafollicular C cells • Elevated serum calcitonin levels • Congo red stain for amyloid and immunohistochemical
stains aid in the diagnosis – Immunohistochemistry:
Positive: calcitonin, CEA, chromogranin, synaptophysin
Negative: thyroglobulin
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Malignant Findings Cytology
• Medullary Thyroid Carcinoma • Isolated, noncohesive cells are the predominant
pattern • Cell clusters can be present • Cells may be many different shapes: 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
Continued on next slide
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Malignant Findings Cytology
• Medullary Thyroid Carcinoma • Binucleation and multinucleation common • Nuclei eccentrically located and typically round to
oval • Spindle cell variant: elongated nuclei • Chromatin is coarsely granular with small nucleoli
(less frequently, prominent nucleoli) • Cytoplasm commonly abundant and finely granular • Nuclear pseudoinclusions may been seen
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Malignant Findings Overview
• Poorly Differentiated Carcinoma • Rare, aggressive malignancy of follicular cell
origin • Can co-exist with a well differentiated
carcinoma (papillary or follicular) and/or undifferentiated (anaplastic) carcinoma
• Patients usually present with advanced disease
continued on next slide
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Malignant Findings Overview
• Poorly Differentiated Carcinoma-continued • Lymph node, lung, and bone metastases
common • Difficult to diagnose by FNA; most often
called “suspicious for a follicular neoplasm” • Immunohistochemistry:
Positive: Thyroglobulin, TTF-1, and cytokeratin Negative: Calcitonin
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• Poorly Differentiated Carcinoma • Markedly cellular • Cells are arranged singly, in crowded
groups, papillary-like aggregates, or microfollicles, and can be wrapped by endothelium, creating insulae or trabeculae
• Naked nuclei, necrosis, and mitoses are often present
continued on next slide
Malignant Findings Cytology
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• Poorly Differentiated Carcinoma -continued • Cytoplasm is scant • 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 with varying sizes of nucleoli
Malignant Findings Cytology
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Malignant Findings Overview
• Undifferentiated (Anaplastic) Carcinoma • Rapidly growing, very aggressive • >age 50, more common in women than
men • Rapid tumor growth infiltrates into
surrounding soft tissues of the neck
continued on next slide
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Malignant Findings Overview
• Undifferentiated (Anaplastic) Carcinoma - continued • Common site for metastasis is the lung • A secondary co-existing thyroid
carcinoma may be present in some cases • Immunocytochemistry:
Positive: Pan-keratin, PAX8 (often positive) Negative: TTF-1 and thyroglobulin (both are usually
negative but can be focally or weakly positive)
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Malignant Findings Cytology
• Undifferentiated (Anaplastic) Carcinoma • Cellularity variable; can be low due to dense fibrotic
stroma and necrosis • Cells are commonly arranged as single cells,
crowded groups, and stripped nuclei • Cells are epithelioid and/or spindle-shaped with
marked variation in size • Giant cells common continued on next slide
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Malignant Findings Cytology
• Undifferentiated (Anaplastic) Carcinoma - continued • 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 • Focal squamous differentiation may be present and
raise the possibility of a squamous cell carcinoma (primary or metastatic)
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Malignant Findings Overview & Cytology
• Squamous Cell Carcinoma • Poor prognosis • Occurs in the elderly • Rare tumor of the thyroid, accounting for <1% of
tumors occurring in the thyroid • Indistinguishable from metastatic squamous cell
carcinoma • Correlate with clinical history and imaging results • Large, pleomorphic keratinized squamous cells
with or without necrosis
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Malignant Findings Types
• Lymphomas • Diffuse large B-Cell lymphoma (DLBCL) • Extranodal marginal zone lymphoma of
mucosa-associated lymphatic tissue (MALT) lymphoma
• Mixed DLBCL and MALT lymphoma
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Malignant Findings Overview
• Lymphomas • Account for 1-5% of all thyroid cancer • Equal split between MALT type, DLBCL and mixed
MALT/DLBCL • Two thirds are preceded by Hashimoto thyroiditis • Mainly affects women in their sixth decade • Rapid onset presenting as a firm, diffuse mass • Can occur in the thyroid either as a primary lesion or
secondarily with the latter being more common • Immunophenotypic subtyping is necessary
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Malignant Findings Cytology
• Diffuse large B cell Lymphoma (DLBCL) • Highly cellular aspirate that is easily
recognizable as malignant • Large, atypical immature lymphoid cells
• Centroblast-like cells – Peripheral nucleoli and scant cytoplasm
• Immunoblasts – Prominent central nucleolus, abundant cytoplasm and
may appear plasmacytoid
continued on next slide
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Malignant Findings Cytology
• Diffuse large B cell Lymphoma (DLBCL) -continued • Lymphoglandular bodies are present • Follicular cells are absent or rare • CD20 and CD45 +, CD5 – • Monotypic light chain restriction • Differential Diagnosis(DDx)-metastatic
carcinoma
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Metastatic B-cell lymphoma
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Malignant Findings Cytology
• Extranodal marginal zone lymphoma of mucosa-associated lymphatic tissue (MALT) lymphoma • (DDx)Hashimoto thyroiditis • Highly cellular aspirate resembling a reactive
lymph node • Heterogeneous population with an increased
number of small to intermediate-size lymphocytes • Immunoblasts, plasma cells, lymphoglandular
bodies and lymphohistiocytic aggregates continued on next slide
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Malignant Findings Cytology
• Extranodal marginal zone lymphoma of mucosa-associated lymphatic tissue (MALT) lymphoma -continued • Absence of cells that transition between intermediate-
size and large lymphocytes • Rare follicular and oncocytic cells are often present • Monocytoid appearance • Light chain monoclonality • Positive for CD20 and CD45, Bcl-2 • Negative for CD5, CD10, CD23, Cyclin D1, Bcl-6
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Malignant Findings Types
• Metastatic Carcinomas and Lymphomas • Metastases are uncommon. Most common primaries:
– Kidney (DDx includes follicular and Hürthle cell neoplasms)
– Colorectal – Lung – Breast – Melanoma (DDx includes medullary and anaplastic
carcinoma) – Lymphoma (DDx includes undifferentiated carcinoma and
Hashimoto thyroiditis) – Squamous cell carcinoma (primary or metastatic)
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Malignant Findings Overview
• Metastatic Carcinomas and Lymphomas • Patient history, flow cytometry (when
considering a lymphoma), and immunocytochemistry (e.g., TTF-1, thyroglobulin) are invaluable for the diagnosis of these cases
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Metastatic Renal Cell Carcinoma
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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
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Bibliography ThinPrep® 2000 Processor Operator’s Manual ThinPrep® 5000 Processor Operator’s Manual Ali, Syed Z., Cibas, Edmund S.: The Bethesda System
for Reporting Thyroid Cytopathology: Definitions, Criteria and Explanatory Notes, 2010.
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: 839-96.
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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 ADS-00892 Rev. 001