EFFUSION CYTOLOGY

NAMRATHAEFFUSION CYTOLOGY

Body cavities - Two layers derived from the embryonal mesenchyme- 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 cellsClear, watery fluid (serous fluid) is produced, which lubricates the organsAny excess of serous fluid is an effusion and indicates a disease state

mesotheliumImage courtesy of Boston Universityperitoneal cavityConnective tissue of serosa

Serous fluid is produced by the serous membranes (typically just a few milliliters) and acts as a lubricant for the organs.

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The outer layer - parietal peritoneum attached to theabdominal walland thepelvic wallsThe inner layer, thevisceral peritoneum- wrapped around the internal organs that are located inside the intraperitoneal spaceThe potential space - (about 50 mL) of slippery serous fluid that allows the two layers to slide freely over each other.

Biological Nature of EffusionAn effusion is any excess amount of serous fluid in a body cavityAlways caused by a pathologic processFluid is reabsorbed after successful treatment.Effusions are classified into four categoriesHydrostaticInfectiousNoninfectious inflammatoryMalignantEach category can be one of two typesTransudateExudate

SAMPLE COLLECTIONThe fluid is collected into a clean, dry container, which need not be sterile, and sent to the laboratory as soon as possible. If the fluid cannot be sent immediately, it should be stored in a refrigerator at 4 degree c and not allowed to freeze. We do not require anticoagulant or fixative to be added to the fluid. Anticoagulation by adding heparin to the receptacle before the fluid is collected does not interfere with cytologic detail.Formalin, alcohol, or any other kind of cellular preservative must not be added to specimens of serous fluid sent to our laboratory. Formalin not only prevents cells from adhering well to a slide but also interferes with the quality of staining by the Papanicolaou method.

METHODS OF PREPARATIONClot on standing- Exudate- High protein content, rich in fibrin.1ml of 5% sodium citrateCentrifuged in a conical tube @ 800rpm for 12 minLavage- 1000rpm- 10min.Smears made from the fluid free sediment.Wet fixed in 95% ethanol for Pap and H & E.Air dried and fixed in methanol for Romanowsky stain.Heavily blood stained- 1% acetic acid.Smears from buffy coat.

Types of staining smearsPAPHx & ECell block for remnant sediment and histopathological examination.Other special stains for the most suspected diseases, to confirm diagnosis.

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Normal young mesothelial cells:

Singly, in doublets, or in clusters of variable sizes and configuration Cohesive, monolayered sheets of cells-mesothelial cells forming small cell balls; may have central collagen core (especially when forcibly removed)The cell groups have lobulated or flower-like borders as compared to the smooth community border typically associated with adenocarcinoma.

Normal Components and FindingsNucleiSingle or binucleatedCentrally located but can be eccentricRound to oval with well-defined, smooth nuclear bordersFine chromatinSmall nucleoliCytoplasmDense center with pale peripheryLacy skirt cell borders the clear outer rim (lacy skirt or halo) corresponds to long, slender, branching microvilli. The cells sometimes have cytoplasmic vacuoles. Two or more mesothelial cells in groups are often separated by a narrow clear zone or window.

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20xCopyright 2012 Hologic, All rights reserved.

Specimen type: peritoneal wash 20xSometimes 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.16

Two cytoplasmic zones can be recognized: a perinuclear, denser zone, and a peripheral, clear zone .The difference is caused by an accumulation of cell organelles in the perinuclear area

Cell clasping phenomenonThe cytoplasm of one cell appears to be clasping another cell. This type of articulation is carried a stage further when the cytoplasm of the clasping cell seems to be about to pinch off the cytoplasm of the cell being clasped.The final scene - appearance of one mesothelial cell within another.

Old mesothelial cells Long standing lesion.Hydropic vacuole in the cytoplasm- signet ring shape.Leucocytes seen.Clusters, acini, papillae with mitoses.

Atypical reactive mesothelial cellsSLE, RAChronic inflammationNeoplasmPulmonary embolism or infarctActive cirrhosis or hepatitisUremiaPancreatitisLong-term dialysisRadiation and chemotherapy

REACTIVE MESOTHELIAL CELLSIncreased numbers of mesothelial cells, singly or in clusters, Varying cell sizesScanty or abundant thick homogenous cytoplasm, Sharp smooth heavy cytoplasmic membranesCentral or eccentric nuclei, Enlarged nuclei with coarsely clumped irregularly distrubuted chromatinRegular sharp nuclear borders, Spherical prominent nucleoli.MultinucleationSibling images are a clue- 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.

Benign Mesothelial cells that mimic cancer cellsBenign Formation MimicsThree Dimensional cells balls, or rosettes AdenocarcinomaPapillae Papillary adenocarcinomaIndian files Breast, small cell carcinomaCell in cell Squamous cell carcinomaSignet ring Breast, stomach cancerSingle cell Lymphoma

Normal ComponentsHistiocytesBean shaped eccentric darkly staining nucleiDelicate foamy cytoplasmUsually present in effusion. Prominent in cancer, TB and embolismLymphocytesTuberculosisLymphoma/Lymphocytic leukemia Efffusions of long duration

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Histiocytes have smaller nuclei than mesothelial cells with granular or vacuolated cytoplasm.

Pleural fluid showing predominantly lymphoid cells with few mesothelial cells and histiocytes

The tuberculous effusionLymphocytes slightly larger than in non tubercular effusionsIndented nucleiThick nuclear membrane and coarse chromatinSmall nucleoliRarely- epithelioid cells, multinucleate giant cells.

Eosinophils

No significance in the first tap.Eosinophilic pleural effusions, defined as a pleural effusion that contains at least 10% eosinophils.Spontaneous pneumothorax.Most eosinophilic effusions are pleural and due to allergic reactions to dust. Drug induced, Pulmonary infarct, pneumonia, trauma, hydatid disease.Loefflers syndrome, Traumatic pericarditis, rheumatic pericariditisAllergic vasculitis, Benign asbestos effusion.

Neutrophils

Presence can have many causes, particularly infection.Color of the fluid: creamy pale yellow (purulent)Pneumonia, TB, Foreign body, abscess, Penetrating traumatic injuries. Malignant effusions are seldom associated with acute inflammation

Plasma cells

TBRAHLMultiple myeloma

Filarial effusions tend to be chylous in nature due to leakage of chyle from the occluded thoracic duct. Exudative effusion may be due to Lymphangitis resulting from incomplete obstruction of lymphatics.

stained wet film of pleural effusion illustrating a scolex of Echinococcus granulosusA stained wet film of pleural effusion -ovum of Paragonimus westermani.

LE Cells

Image reprinted with permission from MLO-Medical Laboratory Observer, November 2008.

(a) Cytospin preparation of the pleural fluid showing plenty of lupus erythematosus (LE) cells (MGG, 400). (b) Cytospin preparation of the pleural fluid showing tart cell (MGG, 400)

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.

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LE cells in the pleural fluid Highly specific for SLE. Neutrophilic phagocytes that contain intracytoplasmic hematoxylin bodies. The hematoxylin bodies are thought to be formed by the opsonization of cells by ANA typically found in SLE patients. In cytological preparations, LE cells must be distinguished from tart cells or pseudo-LE cells, which result from the phagocytosis of nuclear debris by macrophages. The phagocytosed debris within the tart cell is smaller, and has a non-homogenous (clumped) appearance in contrast to the smooth homogenous character of the hematoxylin bodies in true LE cells.The incubation of the pleural fluid at room temperature for several hours may enhance the LE cell phenomenon.

Rheumatoid pleuritisExudate with low glucose and high LDHSmall, unilateral, recurrent.Calcium and cholesterol.Necrotising granulomatous inflammation with central fibrinoid necrosis and surrounding spindle shaped cells.Spindle shaped cells and multinucleate giant cells.Fluffy amorphous granular material in the background, Cytoplasmic inclusionsneutrophils may also contain fat droplets-mesothelial cells are noticeably absent

Benign bone marrow cells including a multinucleate megakaryocyte in extramedullary hematopoiesis secondary to myelofibrosis withanemia.Extramedullary hematopoiesis may be identified in effusions in patients with myelofibrosis. Immature blood cells and megakaryocytes are identified

Pulmonary infarctCellularity ranges from slight to profuseMesothelial cells, macrophages including haemosiderophages, lymphocytes, neutrophils are present, eosinophils may be seen

RBCs- traumatic tap. In hemorrhagic effusions, fresh and degenerated erythrocytes are usually seen against a background of fibrin. Sickle cell anemia may be identified in fluids in the form of sickle-shaped erythrocytes ErythrophagocytosisIngestion of the patient's own erythrocytes by macrophages in pleural or ascitic fluids may be occasionally observed, for example, in dialysis ascites Chediak-Higashi syndrome and some forms of malignant lymphoma Autoimmune hemolytic anemia, induced by Epstein-Barr virus.

Malignant effusionsDirect extension or distant carcinomatous metastasis.Reaction- Increased exudate fluid and stratification of mesothelium.Blockage of lymphatics. Compression of vessels by tumour mass or metastasising tumour cells.Size, shape and number depend on nature of malignant neoplasm.Excellent culture medium- 4-6 weeks- Well preserved - Increased mitosis.Suspect malignancy- Bloody background-Haemorrhagic effusion, lymphocytosis, lipophagocytes.

Large unilateral effusion (>1 litre) in the elderly is highly suspicious for malignancyEffusions associated with malignancies may be just a reactive response without presence of any tumour cells The number of neoplastic cells in recurrent effusions often increases. Ask for re-tap if first tap shows no malignant cells or inconclusive in large suspicious effusions, if initial 4 slides show no malignant cells, re-prep further slides and cell blocks

Abnormal FindingsMetastatic:AdenocarcinomaSquamous Cell CarcinomaNeuroendocrine Tumors Lymphoma/leukemiaMelanomaSarcomaOther Neoplasms

Primary:Malignant MesotheliomaEffusion Lymphoma

To identify cancer cells accuratelyTo identify tumor type and, if possible, the site of primary origin

The Most Common Tumor that Cause Malignant Effusion, by Site and SexType of Malignant Men WomenPleural Lung Breast Gastrointestinal tract Lung Pancreas Ovary Peritoneal Intestinal Ovary (includes gastric and pancreatic) Pancreas Breast Prostate Uterus

Malignant effusions---metastatic tumors

RELATIVE FREQUENCY OF ORIGIN OF METASTATIC MALIGNANT TUMORS IN CHILDREN

Pleural or Peritoneal FluidLeukemia-lymphomaWilms' tumorNeuroblastomaEmbryonal rhabdomyosarcomaEwing's tumor

Comparison - erythrocytes, lymphocytes, or mesothelial cellsLarge or very large

Small

Medium-sized.

Mesotheliomas, metastatic carcinomas of various types, malignant melanomas and sarcomas.Malignant lymphomas, many of the malignant tumors of childhood and certain carcinomas (small-cell carcinoma of the breast, oat cell carcinoma).Mammary, lung, gastric, pancreatic, or prostatic origin may have this presentation

A Few Cytomorphological Patterns Proliferation spheres Breast ca, small cell ca lungIsolated tumour cells Gastric ca, lobular breast ca, melanoma, lymphomaSignet ring cells Gastric ca-Colorectal carcinomaPapillary groups containing psammoma bodies Ovarian ca, thyroid c

Unique to effusion cytologyProliferation of tumour cells in fluid medium (invivo)More common in chronic malignant effusions Breast ca, small cell ca, ovarian caSpheres may fuse to mimic papillary structuresMay also be associated with reactive mesothelialproliferations

Signet-ring CellsMalignant Gastric adenocarcinoma Colonic adenocarcinomaBenign Degenerative vacuoles in mesothelial cellsand macrophages

Psammoma bodies Single cellsMalignant Ovary serous carcinoma Thyroid papillary carcinoma Lung some BAC Mesothelioma papillary epithelial typeBenign Pelvic inflammatory disease Non-specific finding in women in ascitic fluidsand pelvic washing

Malignant Gastric adenocarcinoma (diffuse type) Breast lobular carcinoma LymphomaMelanomaDDx - Benign Reactive lymphoid population

A)psammoma bodies in papillary tumor(B; H&E ), cytoplasmic mucin of signet ring cell carcinoma(C)or cytoplasmic melanin of malignant melanoma(D).(A, C, D; Papanicolaou stain).

Epithelial and mixed mesotheliomas account for about 90% of all pleural and peritoneal primary tumors. Both neoplasms are commonly associated with a serous effusion.Sarcomatous and desmoplastic mesotheliomas are usually not associated with an effusion. 10% of cases the effusions are acellular or contain only rare benign reactive mesothelial cells.Mesothelioma

MesotheliomaAsbestos exposure.Viscid hemorrhagic effusion with increased hyalurinic acid.Overwhelming cellularity.The presence of two distinct cell populations, one benign and the other malignant, as seen in metastatic cancers, is not obviously present.

Single cells, clustered, groups, tissue fragments, monolayered sheets, synctial, papillary, three dimensional balls.True acinar pattern absent.

MESOTHELIOMA Cell to cell apposition with windows.Knobby contours, central nuclei, bi and multinucleation.Pleomorphic, delicate smooth nuclear membrane, finely granular chromatin.Single or multiple macro or micronucleoli and mitosis. Small tumor cell clusters commonly show cell-embracing-cell , "push-in cell junctions and a clear space or window between two adjacent cells.

Thick endoplasm and a fuzzy ectoplasm that is due to the presence of long filamentous microvilli on free cell surfaces.Adenomatoid mesothelioma- extensive cytoplasmic vacuolization, mimicking cells from a mucus-secreting adenocarcinoma or signet-ring cell carcinoma.

Smear of pleural effusion depicting mesothelioma cell with their typical intercellular articulation, including the clasping type of articulation

Individual malignant mesothelial cells exhibit a rim of ruffled, less dense cytoplasm (ectoplasm), surrounding dense cytoplasm around the nucleus (endoplasm).

PK-like cells in effusion specimens was highly specific for the diagnosis of MM, especially when the PK-like cells were moderate or numerous

Suggested Immunochemistry MarkersReactive mesothelial cellsCalretinin+CK 5/6 +p53-Desmin+EMA-

MesotheliomaCalretinin+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. 62

DDxReactive mesothelial hyperplasiaMetastatic adenocarcinomaSerous papillary tumour of peritoneum.Sqaumous cell carcinoma.Malignant melanoma.

METASTATIC CANCERS45% in pleural fluid, 27% in pericardial and 35% in peritoneal fluids.More frequently epithelial malignancies.Most adenocarcinomas, rarely- squamous, small cell carcinomas.Non epithelial- Lymphomas/leukemias, melanoma, germ cell tumours.Soft tissue tumours rarely exfoliate.A known history of cancer, a positive effusion may be the first sign of an unsuspected malignancy. Lung, breast, ovarian, and gastrointestinal cancer. The histologic type of cancer most commonly seen in serous effusions is adenocarcinoma but a variety of other cancers can cause effusions.Less common malignancies are squamous cell carcinoma, small cell carcinoma (SCC), hematopoietic malignancies, melanoma, germ cell tumours and sarcomas.

AdenocarcinomaCytologic features:The most frequent type of neoplastic cells in serous effusionsGlandular acini, papillae or cell balls/cannon ballsSmooth Community bordersIncreased N/C ratiosIrregular nuclear membranesAbnormal chromatinLarge or irregular nucleoliSecretory vacuoles with mucin

Most common cause of malignant effusions.66

Carcinoma breastBreast- Most common malignant effusion in females.Tight clusters, ball like nests.Infiltrating ductal carcinoma- Nuclei are molded against the adjacent cells.Smooth nuclear membrane. Prominent nucleoli. Uniform granular chromatin. Abnormal mitosis. The presence of multiple sex chromatin bodies practically assures the diagnosis of cancer, most likely of mammary origin, regardless of other cell featuresMedullary- Shed singly. Lobular carcinoma- Indian file,pseudo pearl formation. Scanty cytoplasm- DDX- Reactive lymphocytes.Colloid- Mucous containing vacuoles, signet ring cells.Scirrhous- Rows of 4-5 cellsScanty pale blue cytoplasm.

Metastatic carcinoma of the breast. Malignant cells contrasting with benign histiocytes. The classic description of metastatic breast cancer in pleural effusions employs the term "cannonballs" to emphasize the rounded arrangement of tumor cells

Metastatic carcinoma of the breast. Note two separate cell populations: a smaller benign population of mesothelial cells and larger tumor cells. 20X

Smear of pericardial effusion depicting metastatic lobularadenocarcinoma of the breast. All of the cells in this field are small adenocarcinoma cells, some forming a caterpillar-like chain

Lung adenocarcinoma40% of malignant pleural effusions.Medium to large malignant cells.Synctial and papillary fragments with or without acinar pattern.Cytoplasmic vacuoles.Well defined cytoplasm, dense cytoplasm. Large nuclei with high N/C ratio.Giant forms, bizarre nuclei with multinucleation.

Cytologic Differences Between Adenocarcinoma and Mesothelioma Adenocarcinoma Mesothelioma Groupings Community borders irregular knobby outline Windows unusual Windows common

Cells Columnar shape Blebs, skirts

Nucleus Usually eccentric Usually central Pleomorphic and bizarre Less pleomorphic and not bizarre

Cytoplasm Delicate, homogeneous Dense with lacy edges Uniform stain Two-tone staining

Vacuoles Secretory Degenerative

Multinucleated Rare CommonGiant cells

Differences Between Adenocarcinoma and Mesothelioma

Mesothelioma AdenocarcinomaBackground- Inflammatory cells

Cell clasping phenomenonCollagen coresPAS Digestible Alcian blue hyaluronidase digestibleBackground- Inflammatory cells and mesothelial cells

PAS non digestibleAlcian blue hyaluronidase non digestible

Suggested Immunochemistry MarkersMesotheliomaCalretinin+WT-1+CEA-TTF-1-MOC-31-Ber-EP4-B72.3-

AdenocarcinomaCalretinin-WT-1-CEA+TTF-1*+MOC-31+Ber-EP4+B72.3+

*Lung and thyroid only

Electron microscopy may also be needed to differentiate difficult cases.80

Squamous cell carcinomaInfrequently involves serous membranes.Centrally located, do not exfoliate.Metastatic from head and neck and from genital regions.Readily recognised when well differentiated.Isolated cells or whorled, very pleomorphic,pyknotic nucleiThe squamoid character of this of carcinoma - keratinizing squamoid cells with their orange cytoplasm and round or tadpole shapesAnucleated ketain squames and malignant pearls.Central nuclei, abundant dense cytoplasm(DDx- Melanoma)a, well demarcated cell borders.Ectoplasmic/endoplasmic differentiation.(DDx- Mesothelioma)

Smear of pleural effusion depicting a fragment ofnonkeratinizing squamous cell carcinoma of bronchus. Unlike a cluster of mesothelial or adenocarcinoma cells, this cluster of cells has a loose, angulated and untidy appearance (Papanicolaou MP).

Smear of pleural fluid containing a metastatic keratinizing cell of laryngeal squamous cell carcinoma

Delicate concentric lines are visible in the cytoplasm of the larger cell; such lines are evidence of squamoid differentiation (Papanicolaou OL).

Small cell carcinoma and neuroendocrine carcinomaDifferentiation from poorly differentisted carcinoma difficult.Oat cells larger than lymphocytes, Flat mono layerTissue aggregates with moldingHomogenous smudged nuclei, linear cleft between moulded nuclei.Single file arrangements can be seen, Small, rounded cellsInconspicuous nucleoli

Small cell undifferentiated carcinoma, lung origin. Nuclei have small nucleoli and often a salt and pepper look to the chromatin. Cytoplasm is scant. 60x

Leiomyosarcoma. A high-grade uterine leiomyosarcoma metastatic to the lung shows in associated effusion large single pleomorphic malignant cells with bizarre nuclei and dense, granular cytoplas.Biphasic synovial sarcoma may show in associated effusions epithelial-like and spindleshaped tumor cells in loose aggregates. Epithelial-like tumor cells with focal gland-like arrangement may be seen, and a transition between these two types of cells may be observed.

Lymphoproliferative disordersThe malignant lymphomas can be divided into four groups:Large-cell lymphomasSmall-cell lymphomasHodgkin lymphomaMiscellaneous lymphoproliferative and hematologic disorders, including rare types of lymphomas, plasma cell myelomas, and leukemiasCohesive, organized aggregates of cells. Cells of malignant lymphomas do not form associations with each other and the cancer cells lie singly. Although superposition of these cells into thick clusters may occur, it is usually an artifact of preparatory techniquesIn effusions, a malignant tumor characterized by organized cell clusters is not a malignant lymphoma, regardless of the size and make-up of individual cells, although rare exceptions may occur

Marked variation in size, sparse cytoplasm, no vacuoles.Enlarged nuclei, prominent nucleoli, abnormal coarse chromatin, Heavy irregular and indented nuclear membrane, Mitoses.Nuclear fragmentation, previously referred to as karyorrhexis, and now recognized as apoptosis Although this feature is more common in small cell lymphomas it does occur in the large cell variant.CLL- reactive lymphocytosisLarge cell lymphoma- Metastatic carcinomaBurkitt's Lymphoma- Cells of this variant of lymphoma may occasionally be observed in ascitic fluid. - malignant cells of lymphoid type with small nuclear vacuoles, accompanied by large macrophages, some of which were smudged, presumably during smear preparation. Leukemia- Lymphoblastic or myeloblastic

The cells are large with markedly atypical features including large pleomorphic nuclei which may be lobated, one or more prominent nucleoli, and abundant amphophilic cytoplasmm. anaplastic or plasmablastic including a pale area alongside the nucleus or 'hof'. Multinucleated cells may be confused with Reed Sternberg cells. Mitoses are frequent.

Small-Cell Lymphoma

B-cell lymphomas> T-cell lymphomas.Large population of monotonous, small lymphoid cells 6 to 12 m in diameter.Round or oval, with very scanty, barely visible basophilic cytoplasm, nuclei are moderately hyperchromatic, occasionally cleaved or somewhat irregular in shape, but mainly round or oval, small nucleoliSmall irregularities of the nuclear contour and nuclear protrusions may be observed. Granularity of the nuclei, cellules grumeles or lumpy cells, characterized by numerous coarse aggregates of chromatin in otherwise spherical nuclei. Useful fixation artifact that occurred only in malignant disorders, but not in benign lymphocytes.Nuclear fragmentation in the form of massive apoptosis (karyorrhexis) of nuclei may occur and is diagnostic of this group of diseases

lymphoblastic lymphoma Small to medium sized lymphocytes Fine powdery chromatin Scant cytoplasm

Small lymphocytic lymphoma Differential diagnosis: chronic inflammation (tuberculosis)

Isolated cells

Pleomorphic sarcoma Osteosarcoma LiposarcomaLarge and bizarre shapedRound cell sarcoma Rhabdomyosarcoma NeuroblastomaSmall and uniform shapedSpindle cell sarcoma Fibrosarcoma Leiomyosarcoma Synovial sarcoma

SarcomasGerm cell tumours.Malignant melanoma- Macronucleoli.Isolated round cells with prominent nucleoli Fine brown cytoplasmic pigmentation Intranuclear pseudoinclusions Immunocytochemistry stain: S-100(+), HMB-45(+)

Small round cell tumors.Sarcomas.

Up to 50 ml Fluid normally present in peritoneal cavityPeritoneal effusion is called AscitesLaboratory criteria for dividing ascitic fluid into transudate and exudate is not well defined as it is for pleural fluid.Diagnostic peritoneal lavage (DPL) have limited use: 1. Rapid screening for significant abdominal hemorrhage 2. Evaluation of hollow viscus injuries Peritoneal dialysis: submitted to check for infection Peritoneal washing: performed intra operatively to document early intra abdominal spread of gynecologic and gastric Ca.Serosal FluidsPeritoneal Fluid

Peritoneal cytology - Important role in the diagnosis and staging of abdominal and gynecologic neoplasms. Peritoneal involvement by malignant tumors - extensive - mesothelioma or high-grade abdominal or ovarian carcinomas, diagnosis can usually be confirmed by examination of ascites.This may include examination of peritoneal washings, brushings, or smears.

Cytologic sampling of fluid from the peritoneal cavity at the time of surgery - ovarian tumors by Keettel and Pixley (1958). Purpose - Improve the staging of these tumors. In 1958, Keettel and Pixley - Procedure may provide evidence of spread of ovarian cancer in the absence of visible lesions. In 1986 - Incorporated into the official staging of ovarian cancer by the International Federation of Gynecology and Obstetrics (FIGO).The presence of cancer cells modifies the staging of ovarian tumors from stages Ia or Ib to Ic and from IIa and IIb to IIc. The higher staging calls for a different approach to treatment with the recognition that surgery alone is not likely to be curative of the disease

PFC in Staging of Gynaecological Cancers 1988 FIGO staging for ovarian cancers requires PFC analysis (1C and 2C) 2009 FIGO staging for endometrial cancers does not require PFC analysis Useful to record findings in histology reports if possible

INDICATIONS AND GOALS OF PERITONEAL EFFUSION ANALYSIS

1. To assist in the initial staging of primary ovarian and endometrial cancers.2. To exclude intra abdominal occult cancers in patients undergoing surgery for benign pelvic diseases.3. To monitor the effectiveness of chemotherapy for advanced ovarian cancers (second-look laparotomy).4. To assist in staging of some non-gynecologic abdominal cancers (gastric and pancreas)

Second-look laparotomy

Patients who had chemotherapy or radiotherapy for advanced ovarian cancer and show no clinical or diagnostic imaging evidence of residual tumor may undergo a laparotomy to evaluate the response to therapy. In about 50% of cases residual tumor is identified grossly, and in 20% of patients an evidence of residual tumor is confirmed by tissue biopsy and/or PW. PW is this situation has a low sensitivity, ranging between 31 and 86%, according to several reported series

Ovarian neoplasms, - positive peritoneal washing will upstage the diagnosis to a FIGO Stage Figo IC. The prognostic significance of malignant cells in washings or brushings varies, depending on the primary ovarian lesion, as for example in serous borderline tumor or serous papillary carcinoma. Positive cytology is frequently a reflection of adnexal involvement in endometrial carcinoma.Carcinoma of the cervix but when identified- Aadvanced disease and may require endocavitary chemotherapy to prevent recurrence.

Positive peritoneal washings - Peritoneal recurrence and poor prognosis in patients with gastric adenocarcinoma.In potentially resectable pancreatic carcinoma-positive cytology is associated with more advanced disease

PFC in Gynaecological Cancers Saline (salt solution) is introduced into the peritoneal cavity, and then removed by suction. After this, the fluid is examined for malignant (cancer) cells (called peritoneal washing cytology).Extensive - mesothelioma or high-grade abdominal or ovarian carcinomas, diagnosis can usually be confirmed by examination of ascites.Additional procedures to assist diagnosis are required for localized tumors, less advanced disease, or follow-up of treatment. This may include examination of peritoneal washings, brushings, or smears. Ascites/effusions - diagnostic Washings/lavage - staging

SAMPLE COLLECTIONPeritoneal lavage cytology specimens may be obtained as an outpatient procedure using a temporary dialysis catheter for patients with ovarian cancer undergoing endocavitary chemotherapy..Implantable intraperitoneal chemotherapy port systems can be used as sampling devices to collect washings for detection of persistent intraperitoneal disease in ovarian carcinoma.

Normal Peritoneal WashingPeritoneal washings and brushings differ from effusions with detached sheets of mesothelial cells, which may give rise to problems in interpretationCellular with large sheets of mesothelial cells that are forcely exfoliated by washing. Intensely bloody, fat and debrisSingle and clustered histiocytes, polymorphonuclear leukocytes - contaminated blood. Cohesive polygonal mesothelial cells with oval nuclei, thin and regular nuclear contours, fine chromatin and small nucleoli. On rare occasions papillary clusters of benign mesothelial cells containing psammoma bodies and masses of collagen surrounded by mesothelial cells (collagen balls).

A large and irregular sheet of mesothelial cells forcely exfoliated by peritonea jet washing showing evenly spaced mesothelial cells that have ill-defined cytoplasm oval or bean-shaped nuclei, small conspicuous nucleoli

Reactive cells in ascitis fluidCyst or adnexal torsion, visceral adhesions, organizing hematoma, andInflammatory lesions such as pancreatitis and chronic salpingitis.Intraperitoneal chemotherapy may give rise to cytologic abnormalities that mimic malignancy.

EndosalpingiosisEctopic benign glands lined by tubal-type epithelium. Multicentric and can involve the peritoneum as well as other pelvic structures, including the ovarian surface and paratubal tissue.Low cellularityCohesive aggregates and/or non-branching papillae or clusters of small, uniform cuboidal to columnar cells with scant basophilic cytoplasm, the nuclei display regular nuclear membranes, fine chromatin and small nucleoli. Ciliated or non-ciliated, and unlike malignancy is mitotically inactive. Psammoma bodies , Hemosiderin-laden macrophages, typical of endometriosis, are not usually present. DDx- reactive mesothelium, endometriosis and serous neoplasia. TThe presence of a complex branching architecture, single atypical cells, mitotic activity, nuclear atypia, and prominent nucleoli favors a neoplastic process.

Endosalpingiosis in peritoneal fluid showing a papillary cluster.The round to ovoid nuclei display fine chromatin pattern and occasionalsmall nucleoli. No distinct cilia was noted. Papanicolaou stain..

ENDOMETRIOSISRupture of an endometriotic cyst. Endometrial epithelial cells, endometrial stromal cells and/or hemosiderin-laden macrophages.Small to medium sized ovoid to columnar cells with bland nuclei. sheets or tight balls with a honeycomb pattern, branching tubules. Mild atypia, mainly in the form of distinct nucleoli.Endometrial stromal - histiocytic or spindle cell appearance. When stromal cells are decidualized, they will have more cytoplasm and round to oval nucleiHemosiderin-laden macrophages and hemolyzed blood are usually present in the backgroundDDx - benign hemorrhagic cystic lesions, metastatic endometrial adenocarcinoma, and low grade endometrial stromal sarcom

well-differentiated endometrial adenocarcinoma - loose but usually three-dimensional clusters of cells with coarse chromatin and nuclear membrane irregularities, the N/C ratio may not be increased if the cells have abundant cytoplasm. In endometriosis, however, the cells are low columnar with a bland nuclear chromati and uniform nuclear Membranes.

loose clusters of benign endometrial cells. These cells are of uniform size, small and lack nucleoli with few hemosiderin-laden macrophages

Ciliocytophthoria: detached ciliated tufts of fallopian tube origin. Relatively common in peritoneal specimens from femalesPsammoma bodies These structures appear purple on Pap stain, which is a helpful feature to distinguish them from collagenous balls which stain more green in color15-20% of peritoneal fluid specimens. Mesothelial hyperplasia, endosalpingiosis, ovarian cystoadenoma and adenofibroma. The most common associated malignancy is papillary serous ovarian carcinoma, although these bodies can be infrequently also encountered with endometrioid carcinoma. Thyroid, kidney, lung and rarely mesotheliomaCollagen 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

Collagene ball in peritoneal washing cell film (A) and in cell block (Pap, A x 400; HE, B x 200).

Detached acellular ciliary tufts in this case of endosalpingiosis (Pap stain, 400)

40xCopyright 2012 Hologic, All rights reserved.Pelvic washing 40x: Psammoma body from a case of biopsy confirmed benign mesothelial hyperplasia with psammomatosis.

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FALSE POSITIVE FALSE NEGATIVE-post radiation or chemotherapy - large bizarre multinucleated mesothelial cells-extramedullary haemopoiesis including megakaryocytes-reactive changes from various causes -degenerate benign vacuolated mesothelial cells, mimicking adenocarcinoma

single cell presentation in some carcinomas e.g. breast, stomach, pancreas-bland looking carcinoma e.g. renal cell carcinoma, looking like histiocytes-single cell presentation in mesothelioma with only mild nuclear atypia-hypocellular tumours e.g. squamous cell carcinoma, sarcoma-low grade lymphoma-difficult to distinguish borderline ovarian tumours from low grade carcinomas

Ovarian adenocarcinomaMost common source of peritoneal effusion.Evaluation of PW is not required for advanced ovarian cancer but is important for staging stage I or II tumors. A positive PW in these two situations will change the tumor stages to IC and IIC, respectively.

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.117

BORDERLINE TUMOURSThe sensitivity of peritoneal washing as an indicator of peritoneal implants in the literature varies from 69% to 90%.Approximately 30-40% of SBT are associated with positive pelvic washings.Positive pelvic washings are highly indicative of surface ovarian involvement or peritoneal implants. No cytologic features that alone can distinguish invasive from non-invasive serous implants.In SBT there is mild or even absent nuclear atypia and mitoses are infrequent. Psammoma bodies are often present.The presence of tumor cells in washings from a patient with SBT should be interpreted as positive for serous neoplasm (or low grade serous neoplasm), and not reported out as positive for serous carcinoma.

Serous Carcinoma is the commonest histologic type of ovarian cancers Often associated with a positive PW.Papillary clusters, acini, rosettesLarge papillae often have distinct fibrovascular cores. Single, large atypical cells are not commonly present. The neoplastic cells are monotonous, with small hyperchromatic nuclei and multiple nucleoli. Low-grade serous carcinoma - monomorphic, small tumor cells in large tridimensional clusters, Peripheral palisading of nucleiPsammoma bodies are often present within tumor cell clusters from low- as well as high-grade tumors

In the mucinous cystadenoma, the epithelial component recapitulate that of the endocervix and produces abundant mucin. The mucinous cells are columnar, with small, round, bland nuclei and lack cilia Mucin is commonly seen in the backgroundof appropriately prepared cytologic materia

Mucinous Carcinoma of the ovary Less common than serous carcinoma.

Most ovarian mucinous tumors are of intestinal type (85%) and present as unilateral tumors. The other histologic subtype, endocervical-like mucinous carcinomas are bilateral in up to 40% of cases. Regardless of their subtypes, these ovarian tumors yield in PW malignant cells with large cytoplasmic vacuoles arranged in loose clusters and in linear arrangements or cell strips or singly

In serous tumors, papillary groups, psammoma bodies, and clear to basophilic granular cytoplasm with rare vacuoles are the cytologic hallmarks.In mucinous tumors, extracellular mucin, intracytoplasmic vacuoles, and a foamy cytoplasm can be helpful cytologic clues to the diagnosis.

Key features of pseudomyxoma peritonei Difficult to aspirate from peritoneal cavity; Mucin with occasional gland cells, isolated or inclustersor strips; and Cells non-atypical (adenomatous) or atypical(carcinomatous).

Key features of immature teratoma Immature cells, usually neuroblastic tissue, are key. Small cells with high N/C ratio and primitive chromatin. Differentiated elements may also be present as in mature teratomas.Key features of dysgerminoma Large cells with variably-sized nuclei;. Prominent nucleoli and foamy cytoplasm; and lymphocytes in the background.

Choricarcinoma in a pelvic washing. A multinucleate syncytiotrophoblast is present in a bloody background (Pap stain, 400)

Key features of adult granulosa cell tumor Highly cellular; Granulosa cells arranged in cords and rosette formation; Many single cells; and Prominent nucleoli and nuclear grooves.

Carcinoma endometriumEndometrialPapillary clusters or single malignant cellsDelicate cytoplasm that is scant to abundantCoarse chromatinMacronucleoliPsammoma bodies may be seen

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Peritoneal effusion - Malignant cells clustered around psammoma bodies.

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Cervical cancerThe incidence of positive PW in cervical cancer of all stages is low, about 8%, and it is about 1% in stage IB cervical cancer. PW finding is not included in FIGO staging system for cervical cancer. Adenocarcinoma of the cervix more commonly spreads to the peritoneal cavity than its squamous cell counterpart. Adenocarcinoma cells are seen in clusters, as well as those of a non-keratinizing squamous cell carcinoma. Cells derived from a keratizing squamous cell carcinoma are often present singly.

Specific Patterns of AdenocarcinomaGastricIntestinal type sheds clusters of large, highly atypical cellsGastric type sheds single signet ring cellsColorectalPapillary or acinar aggregates of tall columnar cellsPalisading nuclei with highly irregular nuclear bordersSignet 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.131

Malignant glandular cells arranged in a dense, rounded cluster. Note that the surface cells maintain cell polarity with apical cytoplasm. The nuclei are irregular with prominent nucleoli.

Cells with malignant features are present as a distinct population. Some may exhibit nuclear displacement by a large secretory vacuole, a "signet ring" cell.

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RenalPapillary or acinar groups of cellsGranular or clear cytoplasm (dense in effusions)

Pleural fluid: Renal cell carcinoma133

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

Peritoneal wash Metastatic pancreatic adenocarcinoma

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10-15 ml fluid normally present in pericardial spaceCauses of pericardial effusion: 1)infection 2)neoplasm 3)MI 4)hemorrhage 5)methabolic 6)RAHIV infected patients commonly have asymptomatic pericardial effusion In HIV associated cardiac temponade 45% are idiopathic, Tb and bacterial infections each accounts for 20% of cases Large effusions (>350 ml) most often caused by malignancy or uremiaBlood-like fluid represent hemorrhagic effusion or aspiration of blood from the heartHct comparable to peripheral and blood gas analysis help to differentiate Serosal FluidsPericardial Fluid

Bronchoalveolar Lavage

1. IndicationsFor the detection and characterization of microbiologic pathogens (primarily Pneumocystis carinii, viral, fungal and bacterial) in immunocompromised patients; for detection and characterization of malignancy.Diffuse disease processInterstitial lung diseaseLymphoproliferative disordersMalignancy

2. Specimen RequiredBronchoscopically obtained lavage (preferably at least 20 mL) of the distal airways and alveoli in the distribution of the suspected lesion.

BAL cytologyAdequacy: abundant alveolar macrophagesInadequacy: bronchial epithelial cells or squamous cells >75%Normal compnent

40xCiliated Columnar Epithelial CellsCopyright 2012 Hologic, All rights reserved.Nuclei: - Basally oriented - Round to oval with smooth nuclear membranes - May contain a nucleolus - Fine to mildly coarse chromatin - Variable in sizeCytoplasm: - Homogenous and basophilic

Group of benign ciliated glandular bronchial cells in a bronchial washing. 138

Goblet Cells

60xCopyright 2012 Hologic, All rights reserved.Bronchial washing: Goblet cells have abundant, finely vacuolated, slightly basophilic, delicate cytoplasm that is filled with mucin. Nuclei are uniform and basally located. Stripped nuclei are common due to the degenerative nature of these cells

Bronchial washing: Goblet cells have abundant, finely vacuolated, slightly basophilic, delicate cytoplasm that is filled with mucin. Nuclei are uniform and basally located. Stripped nuclei are common due to the degenerative nature of these cells.139

60x60xCopyright 2012 Hologic, All rights reservedCopyright 2012 Hologic, All rights reservedBronchial washing: Metaplasia cells are uniform in size and shape. They appear in cobblestone arrangements or loose sheets, cytoplasm is dense with distinct cellular borders. Nuclei are round, with granular to coarse or pyknotic chromatin. Nucleoli are only present if the cells are irritated or reactive.

Bronchial washing: Metaplasia cells are uniform in size and shape. They appear in cobblestone arrangements or loose sheets, cytoplasm is dense with distinct cellular borders. Nuclei are round, with granular to coarse or pyknotic chromatin. Nucleoli are only present if the cells are irritated or reactive. 140

Normal Components and FindingsClara cells- Non-ciliated bronchiolar cells- Secrete a protein that acts as a clarificant, a function similar to mucusKulchitsky cells- Scattered basal epithelial cells- Contain neurosecretory granules- Parent cells of carcinoid tumors

Clara cells are more numerous deeper in the bronchi whereas goblet cells decrease in number.141

MacrophagesCarbon-laden

Hemosiderin-laden

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Carbon histiocytes are more common in smokers, urban dwellers, and in anthracosis (Coal Miners disease); also known as Dust cells Hemosiderin-laden macrophages may be present due to infarcts, heart failure, hemosiderosis or malignancy142