ThinPrep® Non-Gyn Lecture Series

ThinPrep® Non-Gyn Lecture Series
Body Fluid Cytology

Benefits of ThinPrep® Technology
The use of ThinPrep Non-Gyn for body fluid specimens: Optimizes cell preservation Standardizes specimen preparation Simplifies slide screening 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 and saline for troubleshooting These items are those that you are likely to already have in your laboratory.

Recommended Collection Media
CytoLyt® Plasma-Lyte® Polysol® Balanced electrolyte solutions These solutions are key in producing optimal results.

Non-recommended Collection Media
Normal Saline Culture Media RPMI 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®
Hologic has developed these two preservative solutions to work with the ThinPrep® Processors as a complete system. We’ll now discuss their specifications and usage. Copyright © 2012 Hologic, 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 Non-Gyn 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

Sample Collection Fluid Specimens
Concentrate fresh sample by centrifugation before adding CytoLyt® Solution If not possible, collect samples directly into CytoLyt Solution The minimum CytoLyt to sample ratio should be 1:3 Included in “fluids”: Body Cavity Fluids, Cerebrospinal Fluid (CSF), cyst fluids. Fluids collected into CytoLyt require a wash prior to processing on the T2 or T5. We’ll talk about the procedure for this later.

Sample Preparation Sample collection
Concentrate by centrifugation 600g for 10 min - alternately you may centrifuge 1200g for 5 min Pour off supernatant and vortex to resuspend cell pellet Add 30mls of CytoLyt® Solution. Repeat centrifugation, pour off supernatant Evaluate cell pellet . If cell pellet is not free of blood, do a CytoLyt wash and repeat steps 2-4 Add recommended # of drops of specimen to PreservCyt® Solution Vial Allow to stand for 15 minutes Prepare slide on ThinPrep® 2000 using Sequence 2 or ThinPrep 5000 using Sequence Non-gyn Fix, Stain, and Evaluate As stated in previous slide; it is best to concentrate fresh fluid before the addition of CytoLyt. One CytoLyt wash is necessary, but up to two may be done if pellet is not free of blood.

Sample Collection Fresh – recommended
-For extremely bloody fluids, start processing with only 10 ml of fresh fluid CytoLyt® -If fresh collection is not possible, collect samples directly into CytoLyt Solution -The minimum CytoLyt to sample ratio should be 1:3 and is not considered a wash step, but only a collection step. A CytoLyt wash is required prior to instrument processing

Sample Preparation Techniques
Centrifugation-600g for 10 minutes or 1200g for 5 minutes - Concentrate cellular material in order to separate the cellular components from the supernatant Refer to Centrifuge Speed Chart in the ThinPrep® 2000 or ThinPrep 5000 Owner’s Manual, Non-Gynecologic section to determine the correct speed for your centrifuge to obtain force of 600g or 1200g Can also centrifuge at 1200g for 5 minutes---helpful when processing bloody fluids that require 2 CytoLyt washes.

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 - Randomize the cell pellet and to improve 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 a cell pellet, concentrate by centrifugation, pour off the supernatant, vortex and evaluate cell pellet One CytoLyt wash is necessary, but up to two may be done if pellet is not free of blood

Evaluate cell pellet If cell pellet is white, pale pink, tan or not visible, calculate number of drops of specimen to be added to the PreservCyt® Solution Vial (will be discussed in detail on future slides) If cell pellet is distinctly red or brown indicating the presence of remaining blood, conduct a second CytoLyt® Wash. As previously discussed, to conduct a CytoLyt Wash, add 30ml of CytoLyt Solution to specimen, concentrate by centrifugation, pour off supernatant, vortex to resuspend cell pellet. While one CytoLyt wash is necessary, up to two may be performed.

Calculate how many drops of specimen to add to PreservCyt® vial: - If pellet volume is > 1ml (if not consider next 2 slides) 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. Hologic recommends using standard, 1ml, graduated, plastic pipettes.

Calculate how many drops of specimen to add to PreservCyt® vial: - If pellet is clearly visible and pellet volume is < 1ml (if not consider next slide) • 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. Hologic recommends using standard, 1ml, graduated, plastic pipettes.

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

Sample Preparation Troubleshooting
Due to the biological variability among samples and variability in collection methods, standard processes may not always yield a satisfactory and uniformly distributed preparation on the first slide.

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.

No, continue to next slide
Troubleshooting Bloody or Proteinaceous Specimens Check to see if cellularity is adequate. If not, use more of the pellet, if available and process new slide. “Sample is Dilute” message Yes “Yes”: Check to see if cellularity is adequate. If not, use more of the pellet if available. 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

No, continue to next slide
Troubleshooting Bloody or Proteinaceous Specimens Dilute 20:1 using a calibrated pipette to add 1ml of residual sample to a new PreservCyt® Solution Vial. 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

Bloody or Proteinaceous Specimens
Troubleshooting Bloody or Proteinaceous Specimens Is the slide sparse and does it contain blood, protein or non-cellular debris? Centrifuge remaining specimen from PreservCyt® vial, pour off and vortex. Add 30ml of a 9:1 CytoLyt® to glacial acetic acid solution to the sample, centrifuge, pour off and vortex. Add appropriate number of drops to PreservCyt vial and prepare new slide. If resulting slide is sparse, contact Hologic Technical Service. 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 new slide. No Centrifuge remaining specimen from PreservCyt vial, pour off and vortex. Add 30 ml of saline to sample, centrifuge, pour off and vortex. Add appropriate number of drops 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 of Body Cavities
Body cavities are made of two layers - the visceral layer covers the organs while the parietal layer lines the outer wall. Together they form a self-contained cavity that is lined with mesothelial cells called a serous cavity; specifically, the pleural cavity surrounding the lungs, the pericardial cavity surrounding the heart and the peritoneal cavity surrounding the internal organs of the abdomen.

Histology of the Epithelium
Serous membranes consist of connective tissue that is normally lined by a single layer of mesothelial cells Clear, watery fluid (serous fluid) is produced, which lubricates the organs Any excess of serous fluid is an effusion and indicates a disease state Image courtesy of Boston University Connective tissue of serosa Serous fluid is produced by the serous membranes (typically just a few milliliters) and acts as a lubricant for the organs. mesothelium peritoneal cavity

Biological Nature of Effusion
An effusion is any excess amount of serous fluid in a body cavity Always caused by a pathologic process Fluid is reabsorbed after successful treatment

Effusions are classified into four categories Hydrostatic Infectious Noninfectious inflammatory Malignant Each category can be one of two types Transudate Exudate

Biological Nature of Effusions
Hydrostatic Effusions Due to imbalance of intravascular pressure resulting in increased flow of plasma into body cavities Higher fluid to cell ratio Low protein level Cardiac failure is common cause

Infectious Effusions Due to direct effects of organism or as by-product of inflammation Varying types of inflammatory cells can be present Cultures and/or gram and acid fast stains may be useful to identify an organism High concentrations of lymphocytes and absence of mesothelial cells may indicate tuberculosis

Noninfectious Inflammatory Effusions Due to autoimmune conditions or reaction to a stimulus Autoimmune conditions include rheumatoid arthritis and systemic lupus Reactive conditions include tissue necrosis or radiation therapy

Malignant Effusions Due to a primary tumor (such as mesothelioma) or a metastatic tumor Patient history is extremely helpful in rendering a diagnosis Knowledge of the appearance of the fluid is also helpful (clear, bloody, partially clotted) Clear and non-clotted fluids tend to be benign while cloudy, bloody and/or clotted fluids tend to be malignant.

Transudate Due to physiomechanical factors, i.e. congestive heart failure or cirrhosis Typically clear, pale yellow fluid Low protein content (<3.0 g/dL) Does not clot Low specific gravity (<1.015) Low cellularity Unlikely to contain malignant cells Other causes for the formation of transudates include: nephrotic syndrome, myxedema, peritoneal dialysis, hypoproteinemia resulting from malnutrition, Meig’s syndrome, sarcoidosis and glomerulonephritis.

Exudate Indicates damage to the serous membrane, i.e. inflammation or tumor Usually cloudy, yellow or bloody High protein content (>3.0 g/dL) Tends to clot High specific gravity (>1.015) High cellularity High cellularity can be due to an active inflammatory process affecting the body cavity. Other causes for the formation of exudates include: metastatic tumor, pulmonary infarction, autoimmune diseases, diseases of the GI tract, trauma, certain drugs, radiation therapy, post myocardial infarction, yellow nail syndrome (associated w/malignancy) sarcoidosis, uremia and idiopathic causes.

Additional testing may aide in diagnosis: Glucose – Low in TB, rheumatoid disease and bacterial infections Amylase – High in pancreatitis, esophageal perforation pH – Low in rheumatoid disease, hemothorax, acidosis Ammonia –Intestinal necrosis, perforation, effusion(-) vs. urine(+) Creatinine – Negative in effusion, positive in urine Bile – Included in the differential dx of green appearing effusion Cell Block and Immunochemistry*– Tumor typing Electron Microscopy – Differentiate adenoca vs. mesothelioma Flow Cytometry – Useful in hematopoietic malignancies Low pH in malignancy suggests a poor prognosis. Carcinoembryonic Antigen (CEA) can be slightly elevated in benign conditions including empyema (pus in a body cavity), TB, cirrhosis and pancreatitis. *Immunochemistry panels will be discussed in later slides for specific lesions

Specimen Types Pleural fluid Pericardial fluid
Peritoneal (Ascites) fluid Pelvic/Peritoneal Washing

Pleural Fluid Collected via thoracentesis
Removal of pleural fluid from the pleural space with a needle and syringe Procedure can be used as a sampling technique or to alleviate pressure on the lungs Catheter may be used for larger amounts of fluid or to drain recurrent accumulations The patient is positioned sitting upright with arms raised and supported. A local anesthetic is applied and then the doctor inserts the needle into the pleural cavity and the sample is removed. Benign and malignant conditions can be diagnosed.

Pleural Fluid Sources of benign conditions
Primary pulmonary infection or infarction Secondary due to abdominal disease, i.e. cirrhosis Sources of malignant conditions Most common sources are lung, breast, lymphoid neoplasms, GI and mesothelioma

Pericardial Fluid Collected via pericardiocentesis
Removal of pericardial fluid from the pericardial space using a needle and syringe Procedure can be used as a sampling technique, to relieve pressure on the heart or to administer medication Catheter may be used for larger amounts of fluid or to drain recurrent accumulation Benign and malignant processes detected An intravenous (IV) line may be started and the person may be given medications prior to the sample collection. The patient is positioned lying down. A local anesthetic is applied, then the doctor inserts the needle into the space between the ribs (fifth to sixth intercostals space) on the left side of the chest and into the pericardial sac and removes a fluid sample. An ultrasound may be used to help guide the needle. Sometimes medications will be introduced into the space during the pericardiocentesis, i.e. immunosuppressed patients with infection.

Pericardial Fluid Sources of benign conditions
Congestive heart failure, infection, radiation, hypothyroidism, uremia, hypoproteinemia Sources of malignant conditions Commonly associated with lung or breast, followed by lymphoma, sarcoma and melanoma Infectious etiologies are most common in immunosuppressed patients. Pericardial effusions may contain extremely reactive mesothelial cells. Use caution!

Peritoneal Fluid Collected via paracentesis
Removal of peritoneal fluid using a needle, tubing and a container that may have a vacuum Procedure can be used as a sampling technique as well as a therapeutic technique to alleviate abdominal pressure May need to be repeated periodically with some diseases Volume can be as much as 1 gallon or more Peritoneal effusion is also known as ascites fluid. Causes range from cirrhosis to malignancies. The patient is positioned lying down with the head of the bed raised. A local anesthetic is applied and then the doctor inserts the needle into the abdominal cavity and the sample is removed. A catheter may be used to drain larger fluid amounts or to drain recurrent accumulations.

Peritoneal Fluid Sources of benign conditions
Portal venous hypertension, hypoproteinemia and salt retention Sources of malignant conditions Commonly associated with ovary, breast, GI, lymphoid neoplasms and mesothelioma In malignancies of unknown origin, consider genital tract for women and GI tract for men

Pelvic/Peritoneal Washing
Collected intra-operatively Surgeon irrigates the peritoneal cavity with sterile fluid that is later recollected and evaluated Routinely performed to stage abdominal and pelvic tumors Can also be used to diagnose endometriosis.

Normal Components and Findings
Mesothelial cells in effusions Nuclei Single or binucleated Centrally located but can be eccentric Round to oval with well-defined, smooth nuclear borders Fine chromatin Inconspicuous nucleoli Cytoplasm Dense center with pale periphery Lacy “skirt” cell borders Blunt cytoplasmic processes due to degeneration Mesothelial cells appear the same in pleural fluids, pericardial fluids and peritoneal fluids and can be seen singly, in small groups and papillae. A clear space or “window” between adjacent cells is characteristic of mesothelial cells. The cell groups have lobulated or flower-like borders as compared to the smooth “community” border typically associated with adenocarcinoma.

20x Specimen type: pleural fluid 20x
Sheets of benign mesothelial can contain upwards of 50 cells. 20x Copyright © 2012 Hologic, All rights reserved.

40x Specimen type: pleural fluid 40x
Higher magnification of the previous image. 40x Copyright © 2012 Hologic, All rights reserved.

60x Specimen type: peritoneal fluid 60x
Copyright © 2012 Hologic, All rights reserved.

Pericardial effusion: Pericardiocentesis may yield sheets of benign mesothelial cells similar to those found in other body cavity effusions. The etiology of pericardial effusions is more likely to be infectious (viral), autoimmune, inflammatory (myocardial infarction) or reactive (uremia). Tumor, especially lung, may also invade the pericardium. 20x Copyright © 2012 Hologic, All rights reserved.

60x Pericardial effusion 60x: Higher magnification of previous slide.

Normal Components Mesothelial cells in peritoneal washings
Broad and flat sheets Slightly rounded up Cuboidal

20x Specimen type: peritoneal wash 20x
Sometimes sheets fold so that overlapping of cells is evident. The benign nature of these mesothelial cells is obvious based on their uniform arrangement and appearance. 20x Copyright © 2012 Hologic, All rights reserved.

Peritoneal wash 40x Benign mesothelial cells form a peritoneal wash can appear to be rounded up into a more 3D group with some overlap. Closely examine the uniform cells with benign features. 40x Copyright © 2012 Hologic, All rights reserved.

Peritoneal wash 40x Mesothelial cells can also have a cuboidal appearance with more condensed cytoplasm. Note the cohesive sheet of cells with uniformity of nuclei throughout. 40x Copyright © 2012 Hologic, All rights reserved.

20x Pelvic wash: Benign mesothelial cells
A flat sheet of evenly spaced mesothelial cells in a honeycomb pattern, 20x Copyright © 2012 Hologic, All rights reserved.

20x Pelvic wash: Benign mesothelial cells

Pelvic wash: Benign mesothelial cells
Pelvic wash: Benign mesothelial cells. Mesothelial cells can also have a cuboidal appearance with more condensed cytoplasm. Again, note the cohesive sheet of cells with uniformity of nuclei throughout. 40x Copyright © 2012 Hologic, All rights reserved.

Normal Components Histiocytes Lymphocytes Eosinophils Neutrophils
Usually present in effusion. Prominent in cancer, TB and embolism Lymphocytes Some amount usually present. Can be numerous in conditions ranging from congestive heart failure and infections to carcinoma and lymphoma Eosinophils Usually a good prognosis. Most eosinophilic effusions are pleural and due to allergic reactions to dust. Other causes include pulmonary infarct, pneumonia, trauma, hydatid disease Neutrophils Presence can have many causes, particularly infection. Malignant effusions are seldom associated with acute inflammation

Histiocytes have smaller nuclei than mesothelial cells with granular or vacuolated cytoplasm. Compare these histiocytes with the single mesothelial cell at the arrow. 20x Copyright © 2012 Hologic, All rights reserved.

Peritoneal wash 40x: Loose aggregate of histiocytes with delicate, foamy and/or vacuolated cytoplasm, round to oval nuclei with occasional “kidney bean” shapes and irregular chromatin. 40x Copyright © 2012 Hologic, All rights reserved.

Eosinophils on ThinPrep 60x
Eosinophils on ThinPrep 60x. Binucleation and red cytoplasmic granules can be readily identified. 60x Copyright © 2012 Hologic, All rights reserved.

Normal Components Microorganisms LE cells Sickle cells
Charcot-Leyden crystals Psammoma bodies Collagen balls Ciliocytophthoria: detached ciliated tufts of fallopian tube origin. Relatively common in peritoneal specimens from females Microorganisms: almost every type has been described in effusion LE cells: suggestive of systemic lupus erythematosus but can be seen in rheumatoid arthritis and drug reactions as well as in multiple myeloma and Hodgkin’s disease. An LE cell is a neutrophil containing a hematoxylin body, which is a cytoplasmic inclusion of homogenized nuclear material. Look for them in unexplained pleural effusion in a woman of childbearing age. Sickle cells: sickle-shaped RBC’s, can be seen in sickle cell anemia Charcot-Leyden crystals: more likely to form with delayed processing and refrigeration of eosinophillic effusions Psammoma bodies: not diagnostic of malignancy however, ovarian cancer is the most common malignancy with psammoma bodies in an effusion. Also found in benign conditions such as mesothelial hyperplasia and endosalpingiosis. Collagen balls: cores of collagen usually covered with mesothelium. More common in peritoneal washes than in effusions. Can be seen in benign mesothelial proliferations and mesothelioma but are rare in adenocarcinoma

LE Cells LE cell: Wright-stained smear of the pleural fluid shows a typical single LE cell consisting of a neutrophil with flattened nucleus engulfing a central paler homogenous mass. Image reprinted with permission from MLO-Medical Laboratory Observer, November 2008.

Non-cellular Components
Cholesterol crystals Peritoneal wash Starch granules Peritoneal wash Cholesterol and other types of crystals may be seen in peritoneal washes and fluids from other body sites. Starch granules are indicative of glove powder contamination from a prior laparotomy or can be a contaminant from the gloves of the cytoprep tech. 20x 60x Copyright © 2012 Hologic, All rights reserved. Copyright © 2012 Hologic, All rights reserved.

Reactive changes Reactive changes are common and may be due to:
Pulmonary embolism or infarct Active cirrhosis or hepatitis Uremia Pancreatitis Long-term dialysis Radiation and chemotherapy Likely to contain marked inflammation, which is not usually associated with carcinoma. Be cautious when diagnosing malignancy in patients with conditions known to induce marked reactive changes.

Reactive changes Features of reactive mesothelial cells may include:
Varying cell sizes Increased numbers of mesothelial cells Enlarged central or paracentral nuclei Binucleation or multinucleation Coarsening of chromatin that remains evenly distributed Chromocenters and nucleoli may become prominent “Sibling images” are a clue Mesothelial cells in pericardial fluid tend to be highly reactive. Although reactive atypia can be severe, it affects the cells more or less uniformly, resulting in the “sibling image” characteristic of benign cells. In contrast, a secondary “foreign” population of cells usually indicates metastasis.

Pleural effusion 6ox: Reactive mesothelial cells can have a wide range of sizes.

Pleural fluid 60x: Reactive mesothelial cells can exhibit coarse chromatin, irregular nuclear outlines and nucleoli. It is important to look for a spectrum of changes and to correlate the cytology with clinical history. Notice the “windows”. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion 60x: Enlarged nuclei, small multiple nucleoli and spaces “windows” between adjacent cells. Inflammatory cells are present in the background. 60x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion 60x: Mild size variation present in a reactive process.

Pleural fluid 60x: Reactive mesothelial cells tend to present in a more monolayer arrangement and not in the large 3-dimensional clusters of malignant mesothelioma. Again notice the “windows”. 60x Copyright © 2012 Hologic, All rights reserved.

Pelvic washing 60x: The nuclei of mesothelial cells in pelvic washings can exhibit prominent wrinkling, scalloping and grooves possible related to fixation artifact. 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers
Reactive mesothelial cells Mesothelioma Calretinin + CK 5/6 + p53 - Desmin + EMA - Calretinin + CK 5/6 + p53 + Desmin - EMA + Desmin, p53 and EMA can be useful in distinguishing benign from malignant mesothelial cells but this can be variable. Note – Expected staining results; observed in most but not all cases.

Abnormal Findings Tumors causing malignant effusions: Primary:
Malignant Mesothelioma Effusion Lymphoma Metastatic: Adenocarcinoma Squamous Cell Carcinoma Neuroendocrine Tumors Lymphoma/leukemia Melanoma Sarcoma Other Neoplasms

Malignant Mesothelioma
Very rare, usually associated with asbestos exposure Most cases occur in men aged 50 to 70 years, particularly with a background in auto mechanics or construction Commonly found in the pleural cavity but can occur in the peritoneal cavity as well Most commonly located on the right side of the pleural cavity. Heavy and prolonged exposure to asbestos can also lead to peritoneal mesothelioma.

Malignant Mesothelioma
Cytologic features: One cell population of malignant mesothelial cells Larger clusters with irregular, knobby, flower-like borders Dense endoplasm with delicate, lacy ectoplasm “Windows”, “skirts” or blebs of cytoplasm Central enlarged nucleus with macronucleoli and irregular borders Abnormal mitotic figures may be seen but are not diagnostic of mesothelioma Clue to diagnose mesothelioma is “more and bigger cells in more and bigger clusters”. Cell clusters in adenocarcinoma have smooth community borders, with eccentric nuclei and delicate pale to foamy cytoplasm

Pleural effusion (Mesothelioma) - Various magnifications show “cannon-ball” mass of tumor cells. Key to diagnosis is the single cell population. Individual malignant mesothelial cells exhibit rim of ruffled less dense cytoplasm, surrounding dense cytoplasm around the nucleus. Final determination may require cell block with immunohistochemistry or even electron microscopy. . 10x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Mesothelioma) – Higher magnification of the previous image.
40x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion - Tumor cells may be seen in a background of blood and proteinaceous debris. Numerous large clusters of cells may a feature of malignancy. 20x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion – Individual cells exhibit a rim of ruffled, less dense cytoplasm (ectoplasm), surrounding dense cytoplasm around the nucleus (endoplasm). 60x Copyright © 2012 Hologic, All rights reserved.

40x Pleural fluid: One cell population of malignant mesothelial cells

Pleural fluid 60x. The presence of numerous large clusters (>20 cells) in a single cell population of atypical looking mesothelial cells is highly suggestive of malignant mesothelioma. 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers
Mesothelioma Adenocarcinoma Calretinin + WT-1 + CEA - TTF-1 - MOC-31 - Ber-EP4 - B72.3 - Calretinin - WT-1 - CEA + TTF-1* + MOC-31 + Ber-EP4 + B72.3 + Electron microscopy may also be needed to differentiate difficult cases. *Lung and thyroid only Note – Expected staining results; observed in most but not all cases.

Primary Effusion Lymphoma
Very rare subtype of diffuse large B-cell lymphoma Associated with human herpes virus 8 (HHV-8) Pleural, pericardial or peritoneal effusion Most cases occur in HIV positive patients with rare cases in immunocompromised patients Absence of mass lesion and HHV-8 positivity is essential for diagnosis

Primary Effusion Lymphoma
Population of single, large cells Nuclei range from round to irregular and lobulated Coarse chromatin Prominent, irregular nucleoli Cytoplasm is scant to abundant Morphologically similar to diffuse large B-cell lymphoma

Pleural fluid 20x. PEL cells are obviously abnormal even at low power.

Pleural fluid 40x. Cells from this tumor range from immunoblastic (round nucleus with a prominent nucleolus) to anaplastic (large, irregular, multilobulated). 40x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Primary Effusion Lymphoma
CD30 + CD45 + CD5 - CD20 - S100 - CEA - When associated with HIV and HHV-8 infection, these lymphomas are negative for typical B-cell as well as carcinoma and melanoma markers. Note – Expected staining results; observed in most but not all cases.

Adenocarcinoma Cytologic features:
Glandular acini, papillae or cell balls Increased N/C ratios Irregular nuclear membranes Abnormal chromatin Large or irregular nucleoli Secretory vacuoles with mucin Most common cause of malignant effusions.

Specific Patterns of Adenocarcinoma
Breast Cells are usually fairly bland and uniform Aggregates or predominantly single Diagnostic clues are cannonballs, intracytoplasmic lumens and single file chains of cells Positive for mucicarmine stain Breast cancer is the most common cause of malignant pleural effusion in women.

Peritoneal effusion (Breast ca) – Many small clusters and single malignant cells contrast with benign mesothelial cells. As in pleural effusions, these cells tend to be present in large rounded clusters as well as singly. 20x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion (breast ca) – Note the large intracytoplasmic vacuoles with mucin droplets

Pleural effusion (Ductal ca) – A cluster of metastatic ductal carcinoma cells with large irregular nuclei and nucleoli in a background of lymphocytes. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Ductal ca) – Note this single cell expressing nuclear giantism

Pleural fluid – Metastatic lobular carcinoma 60x
Pleural fluid – Metastatic lobular carcinoma 60x. Note the Indian file arrangement of small cells with very high N/C ratios. 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Adenocarcinoma Breast
CK 7 + CK BerEP4 + ER/PR + Mammaglobin + TTF-1 - Note – Expected staining results; observed in most but not all cases.

Lung Large pleomorphic cells that can range from bland to bizarre Nuclei are often hyperchromatic with fine to coarsely granular chromatin and prominent nucleoli - Cytoplasm is often vacuolated and may contain mucin Metastatic lung carcinoma is the most common causes of malignant pleural effusions in men and the second most common cause in women.

Pleural effusion (Lung ca) – Often it is easy to distinguish adenocarcinoma from mesothelioma. Note the eccentric nuclei, lacy cytoplasm and community borders. However, electron microscopy and immunohistochemistry may sometimes be necessary to assist in defining a primary site. Knowing the patient’s history is always the key to identifying metastases. 20x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (lung ca) – Higher magnification of the previous slide.

Pleural effusion (Moderately differentiated lung ca) – Papillary glandular arrangements of tumor cells, prominent nucleoli, vacuolization and mitotic figures. 20x Copyright © 2012 Hologic, All rights reserved.

60x Pleural effusion - Moderately differentiated lung ca

Peritoneal effusion (bronchogenic ca) – cluster of cells with classic features of adenocarcinoma.

60x Peritoneal effusion (bronchogenic ca)

Suggested Immunochemistry Markers – Adenocarcinoma Lung
CK 7 + CK 20 - TTF-1 + BerEP4 + Note – Expected staining results; observed in most but not all cases.

Renal Papillary or acinar groups of cells Granular or clear cytoplasm (dense in effusions) Gastric Intestinal type sheds clusters of large, highly atypical cells Gastric type sheds single signet ring cells Colorectal Papillary or acinar aggregates of tall columnar cells Palisading nuclei with highly irregular nuclear borders Signet rings can also be seen Renal cell cancers are negative for mucin stains. Characteristic features seen are tumor cells with granular centers and clear periphery. Most gastric and colorectal cancers are positive for mucin stains.

Renal Cell Carcinoma The following slides contain examples of renal cell carcinoma

60x Pleural fluid: Renal cell carcinoma. Note the dense cytoplasm.

60x Pleural fluid: Renal cell carcinoma

Pleural fluid 40x: Clear cell type comprises 75% of renal cell carcinomas.

Suggested Immunochemistry Markers – Adenocarcinoma Renal
CK 7 - CK 20 - Vimentin + CD 10 + RCC-m + Staining for clear cell type Note – Expected staining results; observed in most but not all cases.

Gastric Carcinoma The following slides contain examples of gastric carcinoma

Peritoneal effusion (gastric adenoca) – Cells with malignant features are present as a distinct population. Some may exhibit nuclear displacement by a large secretory vacuole, a “signet ring” cell. 60x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion Cell block (gastric adenoca) – The cell block contains cells with similar malignant features as those seen in the previous image from the ThinPrep slide of the effusion. 40x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (gastric ca) – Malignant glandular cells arranged in a dense, rounded cluster. Note that the surface cells maintain cell polarity with apical cytoplasm (the base of the cytoplasm is positioned toward the surface). 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Adenocarcinoma Gastric
CK 7 - CK 20 + MUC5AC + BerEP4 + Adenoca of the stomach shows variable immunochemistry staining patterns. Note – Expected staining results; observed in most but not all cases.

Colorectal Carcinoma The following slides contain examples of colorectal carcinoma

Abdominal wash (colon adenocarcinoma) – This case is from a patient with colon adenocarcinoma. The papillary clusters of cells show obvious features of an adenocarcinoma, but no distinct clues as to the origin of the tumor. 20x Copyright © 2012 Hologic, All rights reserved.

Abdominal wash (colon adenoca) – Higher magnification of the previous image.

60x Abdominal wash (colon adenoca)

Suggested Immunochemistry Markers – Adenocarcinoma Colorectal
CK 7 - CK 20 + CDX2 + Note – Expected staining results; observed in most but not all cases.

Pancreatobiliary Cancers of the pancreas and bile ducts are morphologically indistinguishable Cells may have dense cytoplasm similar to non-small cell lung cancer or vacuolated similar to ovarian cancer Single file chains may be seen

Pancreatobiliary Adenocarcinoma
The following slides contain images of pancreatobiliary adenocarcinoma

60x Peritoneal wash – Metastatic pancreatic adenocarcinoma

Peritoneal effusion – Pancreatobiliary tumors cause obstruction and elicit a hard fibrous host response. Rarely they may liberate cells into the peritoneal cavity, most likely after omental invasion. 20x Copyright © 2012 Hologic, All rights reserved.

60x Peritoneal effusion – higher magnification of the previous image.

Peritoneal effusion – Cholangiocarcinoma, either from an intra-hepatic source or from an extra-hepatic biliary tree, may look like adenocarcinoma from elsewhere in the GI tract. By exclusion of other sources through endoscopy, ultrasonography and/or CT imaging, the location may be determined. 20x Copyright © 2012 Hologic, All rights reserved.

40x Peritoneal effusion - Cholangiocarcinoma

60x Peritoneal effusion - Cholangiocarcinoma

Suggested Immunochemistry Markers – Adenocarcinoma Pancreatobiliary
Pancreatic Adenocarcinoma Cholangiocarcinoma CK 7 - CK 20 + CK17 + MUC1 + CK 7 + CK 20 +/- MUC5AC + Note – Expected staining results; observed in most but not all cases.

Ovarian 3 types Mucinous Serous Endometrioid Most often characterized by cells with large, transparent vacuoles Presence of psammoma bodies may suggest ovarian origin but not diagnostic of malignancy Ovarian cancer is the most common cause of malignant ascites. Differential diagnoses include non-small cell lung cancer, pancreatic carcinoma and mesothelioma. Mucinous type resembles endocervical cells or GI malignancy. Serous type resembles fallopian tubes. Endometrioid type is identical to endometrial carcinoma.

Pleural effusion - Cells of papillary serous ovarian adenocarcinoma in a pleural effusion. Their cell and nuclear size is variable. Increased nuclear to cytoplasmic ratio and cytoplasmic vacuoles are features. Cells may exist singly or in small acinar groups. 20x Copyright © 2012 Hologic, All rights reserved.

60x Pleural effusion– Higher magnification of the previous image.

Pleural effusion - Though three-dimensional, the malignant nature of these ovarian cancer cells is easily appreciated when comparing their size to lymphocytes in the background. Distinction between ovarian and endometrial sources may be difficult given the common Mullerian origin of tumors in each site. 40x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion - Papillary cluster of glandular serous borderline tumor forming rounded “cannon-balls” of tumor cells. Note the smooth border, compared to the scalloped border of reactive mesothelial cells. 60x Copyright © 2012 Hologic, All rights reserved.

Peritoneal wash (Ovarian serous borderline tumor) - Papillary cluster of tumor cells with prominent cytoplasmic vacuolization. Washes are an integral part of staging laparoscopy. Because of the washing procedure, tumor cells generally come off in three- dimensional cohesive groups and may be admixed with sheets of benign mesothelium. The tumor cells are easily distinguished by size, malignant characteristics and crowded configurations. 40x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion - Pleomorphic, papillary groups of tumor cells
Peritoneal effusion - Pleomorphic, papillary groups of tumor cells. Note the size difference between tumor cells and the benign mesothelial cells at lower right. 40x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion - A three-dimensional tumor cell group is seen along with a “dirty” background.

Suggested Immunochemistry Markers – Adenocarcinoma Ovary
Mucinous Adenocarcinoma Non-mucinous Adenocarcinoma CK 7 + CK 20 + BerEP4 + CA 125 + CK 7 + CK 20 - ER/PR + CEA - Note – Expected staining results; observed in most but not all cases.

Endometrial Papillary clusters or single malignant cells Delicate cytoplasm that is scant to abundant Coarse chromatin Macronucleoli Psammoma bodies may be seen

Peritoneal effusion - Malignant cells clustered around psammoma bodies.

40x Peritoneal effusion - Higher magnification of the previous image.

Peritoneal effusion - This photomicrograph shows two separate cell populations. Note the flat sheet at the lower left of uniform, benign cells, many dyshesive tumor cells at lower right, with a large cohesive fragment of tumor cells at upper left. 20x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion - Malignant cells with features of adenocarcinoma including a mitotic figure, prominent nucleoli, vacuolated cytoplasm and eccentric nuclei. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion - Numerous large clusters of malignant cells
Pleural effusion - Numerous large clusters of malignant cells. Note increased cell size relative to lymphocytes and mesothelial cells in the background. 20x Copyright © 2012 Hologic, All rights reserved.

40x Pleural effusion – Higher magnification of the previous image.

Suggested Immunochemistry Markers –Adenocarcinoma Endometrium
CK 7 + CK 20 - Vimentin + B Note – Expected staining results; observed in most but not all cases.

Squamous Cell Carcinoma
Cytologic features: - Large fragments and/or single cells Cytoplasm may be dense and occasionally orangeophilic Hyperchromatic nuclei Coarse chromatin Nucleoli not prominent Primary sites are commonly lung, larynx and female genital tract. Common type of cancer but rarely sheds diagnostic cells into effusions.

Pleural fluid - Note the large, single keratinized cell with coarse chromatin and hyperchromatic nuclei. Contrast this with the cluster of malignant cells in the lower right corner. Cytoplasmic clearing and vacuolization of squamous carcinoma cells can occur in effusions and should not be mistaken for glandular differentiation. 40x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Squamous Carcinoma
CK 5/6 + CEA + p63* + * p63 staining is especially useful in non-keratinizing SCC Note – Expected staining results; observed in most but not all cases.

Small Cell Carcinoma Cytologic features:
- Tissue aggregates with molding Single file arrangements can be seen Small, rounded cells Coarse chromatin Inconspicuous nucleoli Differential diagnoses include breast cancer (small cells, single file chains of cells; look for mucin secretion) and small blue cell tumors of childhood (small cell carcinoma is a tumor of older adult smokers).

Pleural effusion – Small cell undifferentiated carcinoma is an uncommon cause of a malignant pleural effusion. The cells are often in clusters with scant cytoplasm. The nuclei are hyperchromatic with coarse or smudgy chromatin. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion – Cells of small cell undifferentiated carcinoma are dyshesive and can appear singly. Nuclei have inconspicuous nucleoli with “salt and pepper” chromatin. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion – The tumor cells are small and dark, and need to be differentiated from lymphoid cells. Immunohistochemical stains on cell block material may be necessary to confirm the diagnosis. 60x Copyright © 2012 Hologic, All rights reserved.

40x Pleural effusion – Higher magnification of the previous image.

Suggested Immunochemistry Markers – Small Cell Carcinoma
CK 7 + CK TTF-1 + Chromogranin + Synaptophysin + CD56 + Note – Expected staining results; observed in most but not all cases.

Lymphoma/Leukemia Non-Hodgkin Lymphoma is most common and accounts for 10-15% of malignant effusions In children, hematopoietic neoplasms are the primary common cause of a malignant effusion Majority of patients will have biopsy proven disease before effusion develops Reed-Sternberg cells in Hodgkin disease are rarely found in effusions Cytomorphologic features such as cell size and nuclear characteristics (round, irregular, cleaved as well as chromatin pattern and nucleoli) are important in categorizing the lymphoid population. Flow cytometry and immunohistochemistry may be necessary to diagnose lymphoma or leukemia in a patient with no prior clinical history.

Lymphoma/Leukemia Classification of the cells is possible based on:
Size of cells Degree of nuclear abnormalities (cleaved, non-cleaved) Resemblance of cells to lymphoblasts or Burkitt cells Cytologic features: Single cell, monomorphic cell pattern Small cells with scant, delicate cytoplasm High N/C ratios Irregular nuclear membranes (cleaves, nuclear knobs) Prominent nucleoli

Pleural effusion (Non-Hodgkin’s lymphoma) - This pleural effusion also has a distinct population of dyshesive mononuclear cells, allowing the diagnosis of lymphoma. Note the necrotic background. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Non-Hodgkin’s lymphoma) - Cells are most often single or in small loosely adherent clusters. This neoplastic population consists of lymphoid blasts of variable size with prominent nucleoli and irregular nuclear membrane clefts. 20x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Non-Hodgkin’s lymphoma) – High power view of the previous image.

Pleural effusion (Non-Hodgkin’s lymphoma) - These neoplastic lymphocytes appear small and monotonous, but compared to the background lymphocytes, they are considerably larger. Nuclei are vesicular with nuclear clefting and lobation and variably prominent nucleoli. 20x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Non-Hodgkin’s lymphoma) – High power of previous image.

60x Reed-Sternberg cells are much larger than normal cells

Pleural effusion (Chronic lymphocytic leukemia) - The presence of small uniform lymphocytes in a pleural effusion may indicate tuberculosis, thoracic duct obstruction or leukemia/lymphoma. Chronic lymphocytic leukemia and well-differentiated lymphocytic lymphomas may include a pleural effusion consisting of small, round lymphocyte. When a question exists, flow cytometry can confirm the clonal nature of these cells. 60x Copyright © 2012 Hologic, All rights reserved.

Pleural effusion (Chronic lymphocytic leukemia) - An example of small, round lymphocytes in a pleural effusion. In the absence of clinical history, the differential diagnosis includes inflammatory conditions such as tuberculosis, autoimmune diseases and lymphoproliferative disorders. 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Basic Lymphoid Markers
B Cell Markers T Cell Markers CD5 +/- CD20 + LCA (CD45) + BCL-2 + CD3 + CD5 + LCA (CD45) + Hodgkin Lymphoma Markers CD15 + CD30 + LCA (CD45) - Immunochemistry, flow cytometry and molecular studies may all need to be performed to accurately diagnose benign proliferations and various small lymphocytic lymphomas. Note – Expected staining results; observed in most but not all cases.

Malignant Melanoma Cytologic features: Usually single, large cells
Large, eccentric nuclei Frequent intranuclear cytoplasmic invaginations Binucleation is common Very prominent nucleoli Melanin pigment presence is diagnostic Effusion fluid may be dark brown or black. Metastatic melanoma is notorious for its ability to mimic a wide variety of other neoplasms, as well as for late recurrences.

Peritoneal effusion (Malignant melanoma) – May present in effusions with or without an antecedent skin tumor. They often metastasize to unusual places such as small bowel serosa. 20x Copyright © 2012 Hologic, All rights reserved.

Peritoneal effusion (Malignant melanoma) – Cells are dyshesive because they are not true epithelial cells. Nuclei are large and vesicular. Pigmentation of the cytoplasm is often but not always present. In the absence of history of a confirmed skin tumor, immunohistochemistry can confirm the identity of these cells. 60x Copyright © 2012 Hologic, All rights reserved.

Suggested Immunochemistry Markers – Melanoma
HMB45 + Melan-A + MART-1 + Note – Expected staining results; observed in most but not all cases.

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.

For more information… Visit our websites: - Product Catalog - Contact Information - Complete Gynecologic and Non-gynecologic Bibliographies - Cytology Case Presentation and Unknowns - Slide Library Request Form

Bibliography ThinPrep®2000 Operator’s Manual ThinPrep 5000 Operator’s Manual Cibas, E., Ducatman, B.: Cytology: Diagnostic Principles and Clinical Correlates, 3rd edition 2009: DeMay, Richard M: The Art & Science of Cytopathology 1996: DeMay, Richard M.: Practical Principles of Cytopathology 1999: Higgins, et al: Application of Immunohistochemistry in the Diagnosis of Non-Hodgkin and Hodgkin Lymphoma. Arch Pathol Lab Med 2008;132:

Bibliography Koss, Leopold G: Diagnostic Cytology and Its Histopathologic Bases, 4th edition: 1991: Shidham, V., Atkinson, B.: Cytolopathologic Diagnosis of Serous Fluids,

Bibliography Images: LE cell Photograph. NP Communications, Nokomis. Web. (-online.com/features/2008_november/1108_coverstory.aspx) 8 Feb 2012 Mesothelial histology Photograph. Boston University, Boston. Web. 7 Feb 2012 SmartDraw® (Standard Edition) [Software]. (2011). Retrieved from ThinPrep® Non-gyn Morphology Reference Atlas: Body Fluids: 1-49. ADS Rev. 001

ThinPrep® Non-Gyn Lecture Series

Similar presentations

Presentation on theme: "ThinPrep® Non-Gyn Lecture Series"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

ThinPrep® Non-Gyn Lecture Series

Similar presentations

Presentation on theme: "ThinPrep® Non-Gyn Lecture Series"— Presentation transcript:

Similar presentations

About project

Feedback