Cytology of Effusion Fluids Cytology of Effusion Fluids John W. Wong, MD, FRCPC John W. Wong, MD, FRCPC Sunnybrook Health Sciences Centre Sunnybrook Health Sciences Centre Assistant Professor, Laboratory Medicine and Pathobiology Assistant Professor, Laboratory Medicine and Pathobiology Faculty of Medicine, University of Toronto Faculty of Medicine, University of Toronto November 10, 2012 November 10, 2012
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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’
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
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
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
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
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
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
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
–– 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
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
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
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
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
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
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.