Cytology of Effusion FluidsCytology of Effusion Fluids

John W. Wong, MD, FRCPCJohn W. Wong, MD, FRCPCSunnybrook Health Sciences CentreSunnybrook Health Sciences Centre

Assistant Professor, Laboratory Medicine and PathobiologyAssistant Professor, Laboratory Medicine and PathobiologyFaculty of Medicine, University of TorontoFaculty of Medicine, University of Toronto

November 10, 2012November 10, 2012

Cytology of Effusion FluidsCytology of Effusion FluidsLecture ObjectivesLecture Objectives

Clinical importance of malignant effusionsClinical importance of malignant effusionsHandling of effusion specimensHandling of effusion specimensFeatures of benign and malignant effusionsFeatures of benign and malignant effusionsPitfalls in effusion cytologyPitfalls in effusion cytologyUse of ancillary studiesUse of ancillary studiesAccuracy of cytology of effusionsAccuracy of cytology of effusions

AnatomyAnatomy

Four main serosal body cavitiesFour main serosal body cavities–– 2 pleural, 1 pericardial, 1 peritoneal2 pleural, 1 pericardial, 1 peritonealLined by single layer of mesotheliumLined by single layer of mesotheliumNormally contains very little fluidNormally contains very little fluidEffusion develops from imbalance of fluid Effusion develops from imbalance of fluid formation and removalformation and removal

Types of Effusion FluidsTypes of Effusion FluidsTransudateTransudate–– Changes in hydrostatic or osmotic forcesChanges in hydrostatic or osmotic forces–– Ultrafiltrate of plasmaUltrafiltrate of plasma–– S.G. <1.015, protein < 3 g/dLS.G. <1.015, protein < 3 g/dL

ExudateExudate–– Increased capillary permeabilityIncreased capillary permeability–– Rich in protein and inflammatory cellsRich in protein and inflammatory cells–– S.G. >1.015, protein > 3 g/dLS.G. >1.015, protein > 3 g/dL

ChylousChylous–– Rare cause of pleural effusionRare cause of pleural effusion–– Leakage from thoracic duct Leakage from thoracic duct

Causes of EffusionsCauses of Effusions

TransudatesTransudates–– CHF, cirrhosis of liver, nephrotic syndrome, Meigs’ CHF, cirrhosis of liver, nephrotic syndrome, Meigs’

syndromesyndrome

ExudatesExudates–– Malignant neoplasmsMalignant neoplasms–– Infections, collagen vascular diseases, pulmonary Infections, collagen vascular diseases, pulmonary

infarction, post radiotherapyinfarction, post radiotherapy

ChylousChylous–– Malignant neoplasmsMalignant neoplasms–– Trauma, infections Trauma, infections

Sampling of Effusion FluidsSampling of Effusion Fluids

Thoracentesis, paracentesis, pericardiocentesisThoracentesis, paracentesis, pericardiocentesisDiagnostic, therapeutic or bothDiagnostic, therapeutic or bothSample size, a few ml to Sample size, a few ml to litreslitresFor cytology, microbiology, chemistry, cell count, For cytology, microbiology, chemistry, cell count, flow cytometry, other ancillary studies flow cytometry, other ancillary studies

Handling of Effusion FluidsHandling of Effusion Fluids

Fixatives not necessaryFixatives not necessaryCannot do DQ stain if fixedCannot do DQ stain if fixedEffusion fluids act like culture medium; cells will Effusion fluids act like culture medium; cells will remain viable and retain morphology for several remain viable and retain morphology for several days in fridge (4 days in fridge (4 °°C)C)Process fluids promptly to minimize Process fluids promptly to minimize degenerative artifactsdegenerative artifacts

Handling of Effusion FluidsHandling of Effusion Fluids

Gross examinationGross examination–– Volume, colour, opacity, Volume, colour, opacity, odourodour, blood, blood–– Clues to underlying etiologyClues to underlying etiology

SlidesSlides–– Direct smears, Pap or DQDirect smears, Pap or DQ–– LiquidLiquid--based based -- SurePath, ThinPrepSurePath, ThinPrep–– CytospinCytospin

Cell blockCell block–– Clot and/or sediment preparationClot and/or sediment preparation

Question #1Question #1

When do you prepare a cell block for an When do you prepare a cell block for an effusion specimen in your lab?effusion specimen in your lab?

1.1. Routinely for every specimenRoutinely for every specimen2.2. Only if cytology looks suspicious or positiveOnly if cytology looks suspicious or positive3.3. Only if stains are requiredOnly if stains are required4.4. NeverNever

Cytology Cell BlockCytology Cell Block

Various techniquesVarious techniques–– HistoGelHistoGel, agar, gelatin, albumin, , agar, gelatin, albumin,

plasma / thrombinplasma / thrombinAllow special studiesAllow special studies–– IHC, ISH, molecularIHC, ISH, molecular

Significance of Malignant EffusionsSignificance of Malignant Effusions

In patients with known malignancyIn patients with known malignancy–– Indicates advance stage, poorer prognosisIndicates advance stage, poorer prognosisInitial manifestation of malignancyInitial manifestation of malignancy–– Identification of primary siteIdentification of primary siteTreatment implicationsTreatment implications–– palliative versus curative intentpalliative versus curative intent–– Excludes surgery or radiation therapyExcludes surgery or radiation therapy

Malignant Effusions Malignant Effusions -- StatisticsStatistics

Any malignancy may lead to an effusionAny malignancy may lead to an effusionNot all effusions in cancer patients are malignantNot all effusions in cancer patients are malignant

Malignant pleural effusionMalignant pleural effusion–– Men: lung, GI, pancreasMen: lung, GI, pancreas–– Women: breast, lung, ovaryWomen: breast, lung, ovary

Malignant ascitesMalignant ascites–– Men: GI, Pancreas, LungMen: GI, Pancreas, Lung–– Women: Ovary, GI, pancreasWomen: Ovary, GI, pancreas

Approach to Effusion CytologyApproach to Effusion Cytology

Get clinical history, imaging and other Get clinical history, imaging and other laboratory findingslaboratory findingsPresence of malignant cells?Presence of malignant cells?Determine primary site?Determine primary site?Require ancillary studies?Require ancillary studies?Require another sample or biopsy?Require another sample or biopsy?

Benign Cells in EffusionsBenign Cells in Effusions

Mesothelial cellsMesothelial cellsMacrophagesMacrophagesBlood & inflammatory cellsBlood & inflammatory cellsEndometrial cells, tubal cellsEndometrial cells, tubal cellsLiver, colon, lung cellsLiver, colon, lung cells

Benign Mesothelial cellsBenign Mesothelial cells

Spectrum of featuresSpectrum of featuresMonotonous, central nucleusMonotonous, central nucleusNucleoli inconspicuous to prominentNucleoli inconspicuous to prominentTwoTwo--zone cytoplasmzone cytoplasmFuzzy cell borderFuzzy cell borderWindows between cellsWindows between cellsMultinucleation, signetMultinucleation, signet--ring like vacuolesring like vacuolesOccasional papillary groupsOccasional papillary groups

Benign effusionBenign effusion

Reactive Mesothelial cellsReactive Mesothelial cells

Avoid the term “atypical”Avoid the term “atypical”Wide morphologic spectrum overlapping Wide morphologic spectrum overlapping with malignant cellswith malignant cells–– High N/C ratioHigh N/C ratio–– Nuclear hyperchromasia & Nuclear hyperchromasia & pleomorphismpleomorphism–– Coarse chromatin clumpsCoarse chromatin clumps–– Prominent macronucleoliProminent macronucleoli–– Irregular nuclear membraneIrregular nuclear membrane–– Numerous mitotic figuresNumerous mitotic figures

Reactive Mesothelial CellsReactive Mesothelial Cells

Peritoneal WashingPeritoneal Washing

Sampled before operative procedure for Sampled before operative procedure for suspected gynecological malignancysuspected gynecological malignancyUnique cytological featuresUnique cytological featuresFluid is saline not plasmaFluid is saline not plasmaBeware of the specimen labeled as Beware of the specimen labeled as “peritoneal fluid”“peritoneal fluid”

Peritoneal WashingPeritoneal Washing

Benign versus MalignantBenign versus Malignant

““TwoTwo--cell population”cell population”Mesothelial cells show continuum of Mesothelial cells show continuum of morphologymorphologyMalignant cells stand out as distinct Malignant cells stand out as distinct second populationsecond populationIHC if uncertainIHC if uncertain

Mesothelial cell Adenocarcinoma cell

Modified from: Shidham & Atkinson (2006)

BenignBenign–– More single cellsMore single cells–– Cell clustersCell clusters

Knobby, flowerKnobby, flower--likelikeFlat shapeFlat shapeWindowsWindows

–– CiliaCilia–– Degenerative vacuolesDegenerative vacuoles–– Inflammatory cellsInflammatory cells

MalignantMalignant–– Large cell aggregatesLarge cell aggregates

Smooth outlinesSmooth outlinesRounding upRounding upBizarre shapesBizarre shapesDistinct cell bordersDistinct cell borders

–– Secretory vacuolesSecretory vacuoles–– Nuclear pleomorphismNuclear pleomorphism–– Lymphoma, melanoma Lymphoma, melanoma

may show single cellsmay show single cells

Cytomorphological CluesCytomorphological Clues

Malignant EffusionsMalignant EffusionsWhat is the primary site?What is the primary site?

DIAGNOSIS BYORGAN SITE

CYTOMORPHOLOGICAL& ANCILLARY FEATURES

“TEXTBOOK”KNOWLEDGE

CYTOLOGYPRACTICE

Malignant EffusionsMalignant EffusionsWhat is the primary site?What is the primary site?

HistoryHistoryCytomorphological patternsCytomorphological patterns–– Average nuclear sizeAverage nuclear size

S, M, L, GS, M, L, G–– MucinMucin--secreting activitysecreting activity–– Degree of cell cohesionDegree of cell cohesion

Proliferation spheres, papillary structures, Proliferation spheres, papillary structures, cohesive clusters, solitary cellscohesive clusters, solitary cells

–– Specific unique featuresSpecific unique featuresMelanin, psammoma bodiesMelanin, psammoma bodies

Ancillary studiesAncillary studies

Ancillary StudiesAncillary Studies

HistochemistryHistochemistry–– MucinMucin

ImmunocytochemistryImmunocytochemistry–– Typing of tumour Typing of tumour –– epithelial, mesothelial, lymphoid, epithelial, mesothelial, lymphoid,

melanocytic, germ cell, etcmelanocytic, germ cell, etc–– Determine primary site for adenocarcinomaDetermine primary site for adenocarcinoma–– Confirm presence of few tumour cellsConfirm presence of few tumour cells

Flow cytometryFlow cytometry–– Lymphoma / leukemiaLymphoma / leukemia

MolecularMolecular–– Breast, lung, gastricBreast, lung, gastric

A Few Cytomorphological PatternsA Few Cytomorphological Patterns

Proliferation spheresProliferation spheres–– Breast ca, small cell ca lungBreast ca, small cell ca lung

Isolated tumour cellsIsolated tumour cells–– Gastric ca, lobular breast ca, melanoma, lymphomaGastric ca, lobular breast ca, melanoma, lymphoma

Signet ring cellsSignet ring cells–– Gastric caGastric ca

Papillary groups containing psammoma bodiesPapillary groups containing psammoma bodies–– Ovarian ca, thyroid caOvarian ca, thyroid ca

Proliferation SpheresProliferation Spheres

Proliferation SpheresProliferation Spheres

Unique to effusion cytologyUnique to effusion cytologyProliferation of tumour cells in fluid medium (in Proliferation of tumour cells in fluid medium (in vivo)vivo)More common in chronic malignant effusionsMore common in chronic malignant effusions–– Breast ca, small cell ca, ovarian caBreast ca, small cell ca, ovarian ca

Spheres may fuse to mimic papillary structuresSpheres may fuse to mimic papillary structuresMay also be associated with reactive mesothelial May also be associated with reactive mesothelial proliferationsproliferations

Psammoma BodiesPsammoma Bodies

Psammoma BodiesPsammoma Bodies

MalignantMalignant–– Ovary Ovary –– serous carcinomaserous carcinoma–– Thyroid Thyroid –– papillary carcinomapapillary carcinoma–– Lung Lung –– some BACsome BAC–– Mesothelioma Mesothelioma –– papillary epithelial typepapillary epithelial typeBenignBenign–– Pelvic inflammatory diseasePelvic inflammatory disease–– NonNon--specific finding in women in ascitic fluids specific finding in women in ascitic fluids

and pelvic washingsand pelvic washings

SignetSignet--ring Cellsring Cells

SignetSignet--ring Cellsring Cells

MalignantMalignant–– Gastric adenocarcinomaGastric adenocarcinoma–– Colonic adenocarcinomaColonic adenocarcinomaBenignBenign–– Degenerative vacuoles in mesothelial cells Degenerative vacuoles in mesothelial cells

and macrophagesand macrophages

Single Tumour CellsSingle Tumour Cells

Single Tumour CellsSingle Tumour Cells

MalignantMalignant–– Gastric adenocarcinoma (diffuse type)Gastric adenocarcinoma (diffuse type)–– Breast lobular carcinomaBreast lobular carcinoma–– LymphomaLymphoma–– MelanomaMelanomaDDxDDx -- BenignBenign–– Reactive lymphoid populationReactive lymphoid population

ImmunohistochemistryImmunohistochemistry

FormalinFormalin--fixed cell block preferredfixed cell block preferredTry to follow large tumour cell clusters on Try to follow large tumour cell clusters on consecutive levelsconsecutive levelsCell blocks containing mixed populations Cell blocks containing mixed populations of single benign and malignant cells most of single benign and malignant cells most difficult to interpretdifficult to interpretStain panels guided by clinical suspicionStain panels guided by clinical suspicion

Cell Block Stain PatternsCell Block Stain PatternsIsolated Tumour CellsIsolated Tumour Cells

Mostly benign Mostly malignantMixed benign / malignant

ImmunohistochemistryImmunohistochemistryMesothelial markersMesothelial markers–– Calretinin, WT1, CK7, LMWK, CK5/6Calretinin, WT1, CK7, LMWK, CK5/6–– EMA membrane pattern (mesothelioma)EMA membrane pattern (mesothelioma)

Adenocarcinoma markersAdenocarcinoma markers–– CK7, CK20, LMWK, BerEP4, mCK7, CK20, LMWK, BerEP4, m--CEACEA–– EMA cytoplasmic pattern EMA cytoplasmic pattern –– Organ specificOrgan specific

GI tract GI tract –– CDX2CDX2Lung Lung –– TTFTTF--1, Napsin A1, Napsin ABreast Breast –– ER/PRER/PROvarian Ovarian –– ER/PR, WT1, CAER/PR, WT1, CA--125, PAX125, PAX--88Prostate Prostate –– PSA, PSAPPSA, PSAPRenal cell Renal cell –– RCC, CD10, vimentinRCC, CD10, vimentin

ImmunohistochemistryImmunohistochemistrySquamous cellSquamous cell–– HMWK, p63HMWK, p63

MelanomaMelanoma–– HMBHMB--45, Melan A, S100, Sox45, Melan A, S100, Sox--1010

Lymphoma / LeukemiaLymphoma / Leukemia–– CD45, BCD45, B--cell panel, Tcell panel, T--cell panel, etccell panel, etc–– CD30, HHV8, EBVCD30, HHV8, EBV

MacrophageMacrophage–– CD68CD68

Flow cytometryFlow cytometry

Effusions ideally suitable for flow cytometryEffusions ideally suitable for flow cytometrySuspected cases of lymphoma / leukemiaSuspected cases of lymphoma / leukemiaCoordinate with hematology labCoordinate with hematology labMay not be available in community labsMay not be available in community labsRapid transport to flow lab for best resultsRapid transport to flow lab for best results

Case 1Case 1History of pancreatic carcinoma. Ascites.

Case 1 Case 1 -- MacrophagesMacrophages

CD68

CalretininCK7

CB

BerEP4-, mCEA-, EMA-, CK20-, TTF1-, CDX2-

Case 2Case 2Pericardial effusion. Hx of pleural mesothelioma 1 year ago.

Calretinin +

MesotheliomaMesothelioma

Clinical suspicionClinical suspicionPleuritic chest pain, recurrent unilateral Pleuritic chest pain, recurrent unilateral bloody pleural effusionbloody pleural effusionViscous fluid (hyaluronic acid)Viscous fluid (hyaluronic acid)Distinction from benign mesothelial cellsDistinction from benign mesothelial cells–– Bigger clusters, bigger cells Bigger clusters, bigger cells –– Rarely may have only single cells, Rarely may have only single cells,

psammoma bodiespsammoma bodies

ComparisonComparisonBenign mesothelial cells

“Reactive” mesothelial cells

Mesothelioma

Case 3Case 365 M. Pleural effusion. 400 ml cloudy red fluid.

Case 3 Case 3 –– Lung adenocarcinomaLung adenocarcinoma

Calretinin

Napsin ATTF-1

CK7

EMA+, CK20-

Immunohistochemistry Immunohistochemistry –– Napsin ANapsin A

Aspartic proteinaseAspartic proteinasePositive in 80Positive in 80--90% of lung 90% of lung adenocarcinomaadenocarcinomaMore sensitive and specific than TTFMore sensitive and specific than TTF--11Negative in small cell carcinoma, Negative in small cell carcinoma, squamous cell carcinoma and metastatic squamous cell carcinoma and metastatic adenocarcinoma to lungadenocarcinoma to lungGranular cytoplasmic stainingGranular cytoplasmic staining

Case 4Case 478 M. Pleural thickening and effusion. 500 ml red fluid.

Case 4 Case 4 –– Small cell lung carcinomaSmall cell lung carcinoma

Synapto

TTF-1CD56

CB

Case 5Case 542 F. Breast carcinoma. New pleural effusion.

Case Case –– Breast carcinomaBreast carcinoma

ERCB

CK7+, PR+, calretinin-

Case 6Case 688 F. Breast carcinoma. New pleural effusion.

Case 6 Case 6 –– Lobular breast carcinomaLobular breast carcinoma

ERCB

Case 7Case 772 F. Hx breast and colon carcinoma. Ascites 1L cloudy brown.

Case 7 Case 7 –– Colon adenocarcinomaColon adenocarcinoma

CK7

CK20CDX2

CB

ER-

Case 8Case 842 M. Hx gastric carcinoma. Ascites.

Case 8 Case 8 –– SignetSignet--ring carcinomaring carcinoma

PASCB

Case 9Case 953 F. Bilateral ovarian masses. Omental cake. Ascites.

Case 9 Case 9 –– Ovarian carcinomaOvarian carcinoma

Calretinin

PAX-8ER

CK7

CA125+, WT1+, CK20-, CDX2-, TTF1-

Immunohistochemistry Immunohistochemistry –– PAXPAX--88

Transcription factorTranscription factorPositive inPositive in–– MullerianMullerian tract (ovary, endometrium)tract (ovary, endometrium)–– Thyroid, parathyroidThyroid, parathyroid–– Kidney, thymusKidney, thymus–– Some neuroendocrine tumoursSome neuroendocrine tumoursNuclear stainingNuclear stainingStains BStains B--cells (cross reacts with PAXcells (cross reacts with PAX--5)5)

Case 10Case 1053 M. History renal cell carcinoma. Pericardial effusion.

Case 10 Case 10 –– Renal cell carcinomaRenal cell carcinoma

RCCCB

CD10+, calretinin-, TTF1-, CDX2-, PSA-

Case 11Case 1164 F. History melanoma. Pleural effusion.

Case 11 Case 11 -- MelanomaMelanoma

CB S100

HMB45

Panker-, CK7-, CK20-, WT1-, TTF1-

Case 12Case 1234 M. History HIV. KS. Small pleural effusion.

Case 12Case 12

CD3 CD30

EBV-ISHHHV8

CD45+ CD138+ MUM1+ CD20- CD5- ALK1-

Case 12 Case 12 –– Primary Effusion LymphomaPrimary Effusion Lymphoma

DLBCL with primary effusion involvementDLBCL with primary effusion involvementMostly in HIV patientsMostly in HIV patientsMost have HHV8+, some EBV+Most have HHV8+, some EBV+Typical CD45+ CD20Typical CD45+ CD20-- CD30+ CD138+ CD30+ CD138+ HHV8+ EBV+ CD3+/HHV8+ EBV+ CD3+/--Poor prognosisPoor prognosis

Case 13Case 13

48 F mediastinal mass, bilateral lung nodules48 F mediastinal mass, bilateral lung nodulesBiopsy showed “squamous cell carcinoma”Biopsy showed “squamous cell carcinoma”New pericardial effusionNew pericardial effusionSample: 50 ml bloody fluidSample: 50 ml bloody fluid

Case13Case13

Case 13 Case 13 –– Diagnosis?Diagnosis?

1.1. LymphomaLymphoma2.2. AdenocarcinomaAdenocarcinoma3.3. Squamous cell carcinomaSquamous cell carcinoma4.4. Germ cell tumourGerm cell tumour5.5. Do immunohistochemistryDo immunohistochemistry

Case 14Case 14

62 F 15 cm cystic/solid ovarian mass62 F 15 cm cystic/solid ovarian massAscites, bilateral pleural effusionsAscites, bilateral pleural effusionsCT chest CT chest –– enlarged mediastinal nodesenlarged mediastinal nodes700 ml cloudy yellow pleural fluid drained700 ml cloudy yellow pleural fluid drained

Case14Case14

Case 14Case 14

CB calretinin

TTF-1BerEP4

ER- PR- Napsin- PAX8-

Case 14 Case 14 –– Diagnosis?Diagnosis?

1.1. Ovarian adenocarcinomaOvarian adenocarcinoma2.2. Lung adenocarcinomaLung adenocarcinoma3.3. Carcinoma unknown primaryCarcinoma unknown primary4.4. Neuroendocrine carcinomaNeuroendocrine carcinoma5.5. Do more immunohistochemistryDo more immunohistochemistry

Pitfalls in Effusion CytologyPitfalls in Effusion Cytology

Suboptimal specimen preservation or handling Suboptimal specimen preservation or handling may result in degenerative changesmay result in degenerative changes–– Nuclear hyperchromasia, cytoplasmic vacuolizationNuclear hyperchromasia, cytoplasmic vacuolization

The many faces of reactive mesothelial cellsThe many faces of reactive mesothelial cells–– Features overlap with adenocarcinoma cellsFeatures overlap with adenocarcinoma cells

Unexpected patterns or unusual entities Unexpected patterns or unusual entities –– Reactive lymphoid population, single population of Reactive lymphoid population, single population of

pure tumour cells, 3pure tumour cells, 3--dimensional benign cell groups, dimensional benign cell groups, psammoma bodies, megakaryocytespsammoma bodies, megakaryocytes

Effusions Effusions -- Accuracy Accuracy

300 pleural, 300 ascitic fluids300 pleural, 300 ascitic fluidsSensitivity 50%, specificity 97%Sensitivity 50%, specificity 97%PPV 95.7%, NPV 86.4%PPV 95.7%, NPV 86.4%FP 0.5%, FN 31.5%FP 0.5%, FN 31.5%Of FNOf FN–– 30% due to screening error30% due to screening error–– 70% due to sampling error70% due to sampling error

“… diagnostic accuracy of effusion cytology “… diagnostic accuracy of effusion cytology is still unsatisfactory and should be is still unsatisfactory and should be improved.”improved.”

Motherby H et al. Diagn Cytopathol 1999;20:350-7

Closing ThoughtsClosing ThoughtsMany effusions fluids can be reported Many effusions fluids can be reported without ancillary studieswithout ancillary studiesAncillary studies are helpful in determining Ancillary studies are helpful in determining primary site and confirming small primary site and confirming small populations of tumour cellspopulations of tumour cellsAvoid using “atypical” category if possibleAvoid using “atypical” category if possibleBe conservativeBe conservative–– Truly malignant effusions rapidly recurTruly malignant effusions rapidly recur–– Next sample may be more diagnosticNext sample may be more diagnosticTry not be biased by clinical historyTry not be biased by clinical history

BibliographyBibliography

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MotherbyMotherby H et al. Diagnostic accuracy of effusion cytology. Diagnostic H et al. Diagnostic accuracy of effusion cytology. Diagnostic CytopathologyCytopathology 1999;20:3501999;20:350--7.7.