ThinPrep® Non-Gyn Lecture Series

ThinPrep® Non-Gyn Lecture Series
Thyroid Cytology

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

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 These items may be purchased through Hologic.

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 These items are those that you are likely to already have in your laboratory.

Recommended Collection Media
CytoLyt® Balanced electrolyte solutions; such as -Plasma-Lyte® -Polysol®

Non-Recommended Collection Media
Sacomanno and other solutions containing carbowax Alcohol Mucollexx® Culture Media, RPMI Solution PBS Solutions containing formalin These solutions are not optimal for cell preservation and increase the chance for sub-optimal cell morphology.

Hologic® Solutions CytoLyt® PreservCyt®
Copyright © 2012 Hologic, All rights reserved. Hologic has developed these two preservative solutions to work with the ThinPrep Processors as a complete system. Copyright © 2013Hologic, All rights reserved.

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 CytoLyt will preserve morphology of cytology samples for 8 days at room temperature, as long as there is a minimum CytoLyt Solution to sample volume ratio of 1:3.

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

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

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.

Sample Preparation 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 If the specimen has been collected into balanced electrolyte solution (BES), first replace it with 30 ml of CytoLyt.

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

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).

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

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

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 As previously discussed, to conduct a Cytolyt Wash simply add 30ml of Cytolyt Solution to specimen, concentrate by centrifugation, pour off supernatant, vortex to resuspend cell pellet.

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 The type of pipette that you use may affect the concentration of the sample that is added to the PreservCyt Solution Vial, and therefore may affect the volume of sample.

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 The type of pipette that you use may affect the concentration of the sample that is added to the PreservCyt Solution Vial, and therefore may affect the volume of sample.

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 The type of pipette that you use may affect the concentration of the sample that is added to the PreservCyt Solution Vial, and therefore may affect the volume of sample.

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. Troubleshooting is described in further detail in later slides.

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

Techniques Used in Troubleshooting
Diluting the Sample 20 to 1 Glacial Acetic Acid Wash for Blood and Non-Cellular Debris Saline Wash for Protein

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. For uncalibrated plastic pipette make sure to calculate using PreservCyt Solution rather than any other liquid so the drop size will be consistent with the sample drops.

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. This should only be done after a sample has been in PreservCyt Solution. Do not use directly with fresh specimens; nuclear morphology may not be adequately preserved.

Saline Wash for Protein - If sample contains protein, it can be further washed with saline solution in place of CytoLyt® Solution. This should only be done after a sample has been in PreservCyt Solution. Do not use directly with fresh specimens; nuclear morphology may not be adequately preserved.

Bloody or Proteinaceous Specimens
Troubleshooting Bloody or Proteinaceous Specimens Check to see if cellularity is adequate. If not, use more of the pellet, if available and prepare new slide. “Sample is Dilute” message Yes Re-evaluate pellet to determine how many drops of residual pellet are to be added to the PreservCyt specimen vial. Top off vial with PreservCyt to reach a total volume of 30ml. No, continue to next slide

Troubleshooting Bloody or Proteinaceous Specimens 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. Does the slide have a “halo” of cellular material and/or white blood cells? Yes “Yes”: Dilute the sample 20:1. Add 1 ml of sample to a new PreservCyt Solution Vial. No, continue to next slide

Troubleshooting Bloody or Proteinaceous Specimens 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. Is the slide sparse and does it contain blood, protein or non-cellular debris? Yes-blood or non-cellular debris “Yes”: Pour contents of PreservCyt vial into a centrifuge tube. Concentrate by centrifugation. Pour off supernatant and vortex to resuspend cell pellet. If sample contains blood or non-cellular debris, mix 9 parts CytoLyt Solution to 1 part Glacial acetic acid and add 30 ml of this solution to the sample centrifuge tube. If sample contains protein, add 30 ml of saline to the centrifuge tube, concentrate, pour off and resuspend. (Repeat steps if pellet still contains blood or protein.) Add to PreservCyt vial and prepare a new slide. No 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. Yes-protein Contact Hologic® Technical Service

Troubleshooting Common Artifacts
Smudged Nuclear Detail Compression Artifact Staining Artifact Edge of the Cylinder Artifact

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

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

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

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 This may be more evident on highly cellular samples.

Anatomy

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

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)

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

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 in Pap stain and purple in Romanowsky stain

Flame Cells Cytoplasm is abundant and vacuolated-with Romanowsky stain these vacuoles show red to pink staining material which is darker at the edges

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

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

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

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

A fragment of colloid in the center of a follicle shows cracking artifacts at 60x.
Copyright © 2013 Hologic, All rights reserved.

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

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 Note-Specimens with abundant thick colloid don’t require a minimum of follicular cells to be considered Benign and satisfactory. Solid nodules that contain cytologic atypia are never to be considered ND and solid nodules that contain only abundant inflammatory cells should be interpreted as benign as they could arise from Lymphocytic Thyroiditis, thyroid abscess or granulomatous thyroiditis.

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

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

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

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

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

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

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

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

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

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

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

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

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

Benign Findings Benign Follicular Nodule Nodular goiter Colloid nodule
Hyperplasic (adenomatoid) nodule Follicular adenomas (macrofollicular type) Grave’s disease

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

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

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

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

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

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

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

Benign Findings Follicular Adenoma (macrofollicular type)
Most common thyroid neoplasm Almost always occurs as a solitary nodule while the remaining gland is normal

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

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

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

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

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)

Cytology-Atypical Follicular cells of Undetermined Significance
40x 43F Cytology-Atypical Follicular cells of Undetermined Significance Thyroidectomy-Lymphocytic Thyroiditis Copyright © 2013 Hologic, All rights reserved.

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

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

Cytology-Suggestive of a Follicular Neoplasm
40x 48M Cytology-Suggestive of a Follicular Neoplasm Biopsy-Encapsulated Follicular Variant of Papillary Carcinoma Copyright © 2013 Hologic, All rights reserved.

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

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

Isolated tumor cells which present similarly to medullary carcinoma
Isolated tumor cells which present similarly to medullary carcinoma. However, the cytoplasm in Hürthle cell neoplasm is more abundant than in medullary carcinoma. 20x Copyright © 2013 Hologic, All rights reserved.

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

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

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

Malignant Findings Papillary Carcinoma Variants Follicular Cystic
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

Malignant Findings Papillary Carcinoma Variants Warthin-like Oncocytic
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

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

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

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

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)

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

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

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

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

Malignant Findings Poorly Differentiated Carcinoma
Aspirates are highly cellular Cells are arranged singly, in crowded groups, papillary-like 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

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

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)

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

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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Bibliography ThinPrep® 2000 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: 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:

Bibliography Images: Salmon, I. Prof. Normal Thyroid. Photograph. Forpath vzw/asbl, oct; 1999 [ 28 December 2012 SmartDraw® (Standard Edition) [Software]. (2011). Retrieved from ThinPrep® Non-Gyn Morphology Reference Atlas: Thyroid:

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