ser fl cytol ico pune jan 7, 2012 (handout)

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Diagnostic Cytopathology of Effusion Fluids

Short course

International CME on Oncopathology

Diagnostic Cytopathology

Hotel O, Pune, IndiaJanuary 7, 2012

of Effusion FluidsShort course

Vinod B. Shidham, MD, FRCPath, FIACVice-chair & ProfessorVice-chair & ProfessorDirector of Cytopathology, Cytotechnology School, Cytopathology fellowshipDept of Pathology, Wayne State University Medical SchoolDetroit, MI 48201, USA

Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com)

[email protected]

This presentation is available on web for all conference attendees at- http://alturl.com/3ucwx


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DisclosureI do not have conflict of any financial interest in the

product if cited any in the course.

I am the co-editor of the book *and

C th f th h t **Co-author of the chapter**on the same topic.

*Shidham VB and Atkinson BF. Editors and contributors ‘Cytopathologic Diagnosis of Serous Fluids’ Multi-author book with 15 chapters, Elsevier (W. B. Saunders Company), 2007 (ISBN-13: 9781416001454).

** Shidham VB, Falzon M. Serous effusions: reactive, benign and malignant. In Gray & Kocjan, ed. Diagnostic Cytopathology, Elsevier, 3rd edition, Chapter 3.

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International CME on Oncopathology Acknowledgment

Many images and tables are based on chapters in:

Shidham VB and Atkinson BF.Cytopathologic Diagnosis of SerousFluidsElsevier (W. B. Saunders Company) Firstedition, 2007.


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International CME on Oncopathology OutlinePart I :

Anatomy, histology, cytology, and effusions

Collection, transportation, and processing

Factors leading to potential diagnostic pitfalls

Approach to diagnostic cytopathology of effusions

The panorama of different faces of mesothelial cells

Part II :

Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach

Evaluation of unknown primary sites of origin-Where do they come from?

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Anatomy, histology, cytology, and effusions

a. Peritoneal cavityy

b. Left & right pleural cavities

c. Pericardial cavity

Four major serous cavities


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Anatomy, histology, cytology, and effusions (continued)

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Hi t l f li i

32a b

Histology of serous lining(inguinal hernia sac). Themesothelial cells lining thefibrous tissue are flat (1).Focal reactive changes areseen as hypertrophy ofsome cells which assume acuboidal contour (2,3).

dc

( ,3)[a-d, HE stain (a, 10X; b-d,100X)].

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Peripheral light ectoplasm (1) Mesothelial cells (peritoneal


Inner darker endoplasm (2)

Slightly off-center nucleus

Nucleolus

1

fluid): show outer faintly stainedectoplasm (1) with inner denserendoplasm (2) rich inintermediate filaments. Thenucleus is usually central or nearcentral (a), but may be eccentric(b). Nucleoli are readilyobserved The vacuolation1

2

1

2

a b

observed. The vacuolationgenerally begins at the peripheryin ectoplasm (1).[a,b, Papanicolaou stainedSurePath® Preparation (a,b,100XZoomed)]


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Peripheral ectoplasm


Peripheral ectoplasm

Inner endoplasm

Central to slightly eccentric nucleus

Ruffled cell border with blebs

Mesothelial cell (pleural fluid): shows outer ectoplasm which is denser than the innerendoplasm. The nucleus is central to slightly eccentric but not touching the cellperiphery. The cell margin shows blebs and is ruffled.[Diff-Quik stained Cytospin preparation (100XZoomed)]

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Bloody effusions(Hemothorax hemopericardium and hemoperitoneum)


Homogenously red or dark brown with hemosiderin pigment and the hematocrit of the effusion is 10% or more than that of the plasma hematocrit. (Occasional blood tinged fluids may be associated with a procedure trauma). Bloody effusions are more likely to be associated with an underlying malignancy.

Reactive conditions associated with Bloody Effusions

(Hemothorax, hemopericardium and hemoperitoneum)

Para pneumonic effusionsPost traumatic effusions

-Post cardiothoracic procedures / surgeries-Thoracic cavity vascular damage

Pulmonary embolismAsbestos exposure associated pleural effusion

PancreatitisAcute aortic dissectionEndometriosisSarcoidosisIntralobar pulmonary sequestrationSome Infections –e.g B. Anthrax

underlying malignancy


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Biopsy vs effusion cytologyCollection, transportation, and processing

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Collection, transportation, and processing (continued)

To prevent clotting: Collected in anticoagulant such as-EDTA (sodium or potasium salts) in final concentration of 4.55+0.85 µmol/ml[e g di sodium EDTA dihydrate (EDTA Na 2H O) 1 4 mg per ml of effusion fluid][e.g. di-sodium EDTA dihydrate (EDTA Na2, 2H2O) 1.4 mg per ml of effusion fluid]

Amount of fluid sample: Variable (less than 1 ml to 100 ml or more).For optimum cellularity of cytology preparations and cell blocks, it is recommend to submit as

much specimen as possible (up to 1000 ml).Each laboratory follows its own protocol for determining the amount of sample to be used.

Submit as a fresh, unfixed sample:If delay is expected in transporting to the laboratory-

refrigerate at 4oC (with precaution not to freeze the specimen).g ( p p )Alternatively, although not recommended, it may be preserved in a weak fixative

Fluids collected in fixative- require a wash prior to instrument processing.

For blood rich specimens- start with smaller aliquots (such as 10 ml) of fresh fluid.Concentration of cells and removal of excess erythrocytes in the blood rich specimenmay be achieved by density gradient centrifugation with Ficoll


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Concentrate theeffusion fluid specimen

Collect fresh effusion fluid with or without anticoagulant


Concentrate the remaining fluid similar to specimen without clotSpecimen

with clot

Concentrate by centrifugation (at 600g for 10 minutes)

Use homogenized specimen after mixing(For paucicelluar fluids: Use cell-rich sediment)

Direct smear of unconcentrated specimen for semi-quantitative evaluation

Remove the clot and process for

Cell-block preparation Histogel Plasma-Thrombin Celloidin bag

Pour off supernatant and vortex to resuspend cell pellet

Process for paraffin-embedding after formalin fixation.

cell-block preparation

• Direct smears• Cytospin

• SurePrep (Autocyte) • ThinPrep• Other methods

Smear preparation

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Different alternatives available for preparing permanent cytology preparations.a. Direct smears


(Spreading a drop of specimen directly on slide. The specimen may be before concentration OR after concentration as sediment)

i. Wet fixed- Pap stainii. Air-dried-

Diff-Quik stain*Pap stain: After rehydration in saline and

post-fixation in 95%ethanol with 5% acetic acid (28) b. Cytospin preparations (Shandon EZ Megafunnel™ with Shandon Cytospin®) (31)

i. Wet fixed- Pap stainii Ai d i dii. Air-dried-

Diff-Quik stainPap stain: After rehydration in saline and

post-fixation in 95%ethanol with 5% acetic acid (28) c. Filters

i. Wet fixed- Pap staind. Liquid based cytology (SurePathTM or ThinPrepTM or other non-proprietary methods)

i. Wet fixed- Pap stain


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Preparation Purpose


A Diff-Quik stained direct smearof the undiluted specimen

Semiquantitative evaluation of cellularity.

B Diff-Quik stained air-dried Shandon EZ Megafunnel™ preparation

Rapid initial detection of second population and cytomorphologic evaluation of hematopoietic cells.

C Pap stained preparation Cytomorphologic evaluation especially nuclear detailsespecially nuclear details.

D Cell-block preparation in HistoGelTM

Evaluation of-

a. Immunoprofile,

b. Other special stains,

c. Architecture,

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Approach to diagnostic cytopathology of effusions

► GENERAL FEATURES

► PROCESSING RELATED

► INTERPRETATION STRATEGY

► CYTOMORPHOLOGY

► IMMUNOCYTOCHEMISTRY


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Approach to diagnostic cytopathology of effusions (continued)

► GENERAL FEATURESA l h l l b i ff i ll l h l i dAny general pathology laboratory may receive effusion- all general pathologists andcytopathologists should be conversant with the diagnostic challenges and pitfallsof effusion fluid cytology.

►

Finding neoplastic cells in effusion specimens not only reveals that a patient hascancer, but it also denotes the advanced nature of the disease which at this stage isalmost always incurable.

►

The morphologic features of most of the cancer cells in effusion smears arediff t f th i f li ti b hi d FNA t l

►different from those seen in exfoliative, brushing, and FNA cytology.

The standard cytologic criteria of malignancy based on evaluation of single cellmorphology are not applicable for most of the effusion cytology specimens(except in cases with high-grade neoplasms with pleomorphic cells).

►

Since cells in a fluid medium ‘round up’ because of surface tension, the nativeshapes of cancer cells cannot be a guiding feature.

►

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► PROCESSING RELATED


1. Second population of foreign cells

2. Nuclear details of the ‘second population’.

3. Semiquantitative evaluation

4. Objective confirmation and differential diagnosis of primary neoplasm.


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► INTERPRETATION STRATEGY


a. Reactive mesothelial cells- morphologic overlap with neoplastic cellsb. A single population- favor mesothelial cellsc. Second foreign population consistent with metastatic neoplasm d. Sarcomas- previous history knowne. Romanowsky stains highlight the ‘second population’ f. Immunocytochemistry- objective confirmation of ‘second population’y yg. Identical orientation of serial sections for immunocytochemistryh. Quantitative and qualitative clues for mesotheliomai. Apoptosis

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► CYTOMORPHOLOGY


1. Cell groups and intercellular cohesionNon-cohesive, good intercellular cohesion, proliferation spheres

2. Arrangement of neoplastic cellsPapillary configurations

3. Cytoplasm of neoplastic cells

4. Special structures and cytological features

5. Other features


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Algorithm for evaluation of ‘second foreign population’

Reactive-Usually single cells without large 3-D groups

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Cells in effusion fluid 1

Non mesothelial cells 3

Mesothelial cells 2

groups

Hematopoietic cells(Non-cohesive cells)

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Neoplastic-L h

6b

Reactive-Inflammatory cells

6a

Neoplastic- Mesothelioma♦Quantity- Many cells ♦Quality- Many large groups

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Non-mesothelial cells 3 Lymphoma

Neoplastic-8

(2nd foreign population)Carcinoma (Cohesive cells)Sarcoma(Spindle cells may be present. Known history of sarcoma is usually crucial for proper interpretation) Melanoma (Non-cohesive cells)

7 ¶Metastatic cancer cells may be the predominantcells without being seen as a ‘second population’.They may be present as scattered solitary cells withcytomorphology overlapping with floridly reactivemesothelial cells. If indicated, immunocytochemistrywould facilitate confirmation of these cells as non-mesothelial.

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The panorama of different faces of mesothelial cellsREACTIVE MESOTHELIAL CELLS

Binucleation and multinucleation-Binucleation and multinucleationGigantic nucleiPhagocytic activityCohesive Clusters and/or Papillary Structures‘The two cell population approach’Cell-in-cell configurationCells in sheetsGroups of reactive mesothelial cells

I) Hepatomegaly, II) Ischemic conditions such as pulmonary infarction, ischemic colitis, and occlusion of mesenteric blood vessels, III) Trauma to organs covered with mesothelium such as spleen, liver, and lung etc, IV) Large retroperitoneal masses- Slowly growing retroperitoneal masses, V) Postoperative- following laparotomy and thoracotomy.

‘ATYPICAL’ MESOTHELIAL CELLS


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The panorama of different face of mesothelial cells (continued)

a b cMesothelial cells with central to slightly eccentric nuclei (ascitic fluid). The cytoplasm shows a two-zone staining pattern. [a-c, Diff-Quik stained cytospin preparation (a-c, 100x Zoomed)].

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Panorama of mesothelial cells(ascitic fluid). Central to near


a cb

hg

fed

lkji

(asc t c u d) Ce t a to eacentral nuclei. Rare mesothelialcells may show eccentric nucleitouching the cell membrane,but usually there is a narrowrim of cytoplasm separating thenucleus from the cell border(arrows).[ Diff Q ik t i d t i

ponm

xwvuts

rq

[a-x, Diff-Quik stained cytospinpreparations (a-x, 100xZoomed)].


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1


a b c

Mesothelial cells with central to slightly eccentric nuclei (ascitic fluid). The cytoplasm shows a two-zone staining pattern with peripheral vacuolation (red arrow 1). [a-c, Papanicolaou stained ThinPrep preparation (a-c, 100x Zoomed)].

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Mesothelial cells with central tot i l i ( iti fl id)


a cb

hg

fed

l

kji

eccentric nuclei (ascitic fluid).Spectrum of cytomorphologicalfeatures.[a-zc, Papanicolaou stainedThinPrep preparation (a-zc, 100xZoomed)]

ponml

x

wvutsr

q

zy za zb zc


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Mesothelial cells witheccentric nuclei (asciticfluid). Careful scrutinyusually shows a

l x

ynarrow rim ofcytoplasm separatingthe nucleus from thecell border (arrows).[Papanicolaou stained ThinPrep preparation, 100XZoomed].

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NC NCRM


RM

RM

ICIC

DQ Pap

a bMetastatic adenocarcinoma (pleural fluid). An example with morphologically identifiable unequivocal‘second foreign population’ (red arrow NC) other than mesothelial cells (blue arrow RM) and inflammatorycells (brown arrow ‘IC) in DQ and PAP stained preparations. This ‘second population’ of cells ’ (redarrow NC) is easy to be identified in DQ stain (a) as compared to that in PAP stain (b).[a, Diff-Quik stained cytospin preparation; b, Papanicolaou stained ThinPrepTM preparation (a,b, 100xZoomed)]


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a b c da b c dPulmonary adenocarcinoma (pleural fluid). The non-cohesive metastatic cancer cellsis the predominant cell population without being seen as a ‘second population’ (a-d).Some apoptotic tumor cells (arrows in c & d) are present. Differential includesanaplastic large cell lymphoma[a-d: PAP stained Cytospin preparation (a, 10X; b, 40X; c,d, 100X)].

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Factors leading to potential diagnostic pitfallsa. Surface tension related alterations in cytomorphology b Improper specimen processingb. Improper specimen processingc. Many faces of reactive mesothelial cellsd. Proliferation related features

i) Proliferation spheres ii) Increased number of mitotic figuresiii) Prominent nucleoli

e. Degenerative changesNuclear hyperchromasia C l i l iCytoplasmic vacuolation

f. Presence of some unexpected patterns and unusual structuresi) Reactive lymphoid population ii) Polymorphic lymphocytesiii) Single population of cells due to predominance of tumor cellsiv) Psammoma bodiesv) Three dimensional benign cell groups

Benign papillary inclusions, Gland-like epithelial structures, Mullerian inclusionsvi) Megakaryocytes


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Factors leading to potential diagnostic pitfalls (continued)

TRUE NEGATIVE RESULTS IN EFFUSIONS CAUSED BY CANCER

a. Blockage of lymphatics without exfoliation of neoplastic cells

b. Increased capillary permeability due to VEGF.

c L k f f li ti b l d d i dl ll th li

TRUE NEGATIVE RESULTS IN EFFUSIONS CAUSED BY CANCER

c. Lack of exfoliation by low-grade sarcomas and spindle cell mesotheliomas.

d. Organized thick fibrin serosal covering preventing exfoliation of neoplastic cells.

e. Decrease or total disappearance of neoplastic cells over a time

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Part II


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Immunocytochemistry of effusion fluids

The most important issue to be considered when applyingimmunocytochemistry to effusion fluids is the significantvariation in results due to the many variables incurredfrom the time of collection of the specimen to its finalimmunostaining.

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C fi ti f ‘ d f i ’ i fl t l ti f ll th th

UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY


Intricacies of finding and locating the cells of interest in cell-block sections may adversely affect the final results.

►Orient the serial sections identically on all slides

Confirmation of a ‘second-foreign’ non-inflammatory population of cells other than mesothelial cells in effusions correlate with metastatic cancer with objectivity.

It is crucial to:

(to identify more precisely the same cell (or small group of cells) in different sections). ►Know the sequence of these serial sections

(to evaluate their co-ordinate immunoreactivity pattern).

►Immunocytochemistry does not have significant role in evaluation of peritoneal washings.


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Cell-blocks are the preferred choice.


Formalin-fixed cell-block sections are recommended-Other protocols such as the evaluation of various cytology preparations

(direct smears- wet fixed in alcohol or acetone, air-dried fixed with alcohol, air-driedsmears rehydrated and post-fixed in formol alcohol, liquid based cytologypreparations- SurePath or ThinPrep, cytospin preparations, etc) should beavoided.

For reproducible results a standardized protocol with stepscomparable to the processing of formalin-fixed paraffin-embeddedtissue sections is essential.

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Critical issues leading to the best result with cell blocksthe best result with cell blocks

of cytology specimens with singly scattered cells

Aligning the cells along the cutting surfaceAligning the cells along the cutting surface

Monitor the depth of section cutting


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Varsegi GM, Shidham V (2009)Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160

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From:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160


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Modified from:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160


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The video article is available FREE on the web under open access at:

http://www.jove.com/index/Details.stp?ID=1316Vid f J VE ti l (8 i t 15 )

Video of JoVE article (8 minutes 15 sec)

Video of JoVE article (8 minutes 15 sec)


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Immunophenotyping and cell blocks-Factors affecting immunoreactivity-Factors affecting immunoreactivity-

Loss, reduction, or enhancement of antigen immunoreactivityExposure to different reagents and fixative(s)TemperatureStorage of specimen with or without fixative

Subtractive Coordinate Immunoreactivity Pattern (SCIP) approachShidham & AtkinsonCh 5 Imm noc tochemistr of eff sion fl ids introd ction to SCIP approachCh 5. Immunocytochemistry of effusion fl uids: introduction to SCIP approach. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)

Shidham VB.Effusion Fluid Evaluation Made Simple: A brief review of cytomorphologic and SCIP approachCytoJournal (In press).

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Discrepant results between formalin-fixed paraffin-embedded tissue sections of surgical pathology material and effusion fluid cell-block sections are not uncommon


surgical pathology material and effusion fluid cell block sections are not uncommon.

Reasons for variable reports:

The variables responsible for such discrepancies include: sample size (tiny cell groups or single cells), selection of fixatives, antigen retrieval methods

(i e heat induced epitope retrieval enzyme digestion etc )

The proteinaceous effusion fluid around suspended cells may contribute to unexpected nonspecific immunoreactivity.

(i.e., heat-induced epitope retrieval, enzyme digestion, etc.), antibody clones used, antibody titer, and other variations in immunostaining protocols.


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If findings are equivocal:


It is prudent to be conservative and recommend to repeat.Malignant effusions usually re-accumulate quickly.Acquiring a new sample is generally not a problem.

However, it is not uncommon to submit only a small fraction of a large volume ofeffusion fluid collected.

To avoid inadequate resubmission, it may be specifically communicated in therecommendation as comment :

“Recommend submission of most of the drained effusion fluid (up to1000 mL). Larger volume of specimen facilitates retrieval of adequatecellular material in cell-block sections for immunocytochemicalevaluation”.

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I t t ti


All aspects of individual and complimentary immunomarkersshould be considered collectively rather than applying areflexive positive-negative approach.

Evaluation of co-ordinate immunoreactivity in different cell

Interpretation:

ypopulation.


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MicrovillousMembranousCytoplasmicNuclear &NuclearNoneImmunostaining


WT-1

Vimentin

Cytokeratin*

D2-40 (Podoplanin)

Calretinin

MicrovillousMembranousCytoplasmicNuclear & cytoplasmic

NuclearNoneImmunostaining

pattern

Immunomarker

X

X

X

X

X

X

X

PGM1 (CD68)

HBME-1

EMA

LCA (CD45)X

X AdCa

X AdCa

X

X meso

X meso

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MicrovillousMembranousCytoplasmicNuclear &NuclearNoneImmunostaining


MicrovillousMembranousCytoplasmicNuclear & cytoplasmic

NuclearNoneImmunostaining

pattern

Immunomarker

MOC-31

BerEP4

Cadherins

CD44S

B72.3 X

X

X

X

X

CA19.9

Ttf-1

Mesothelin

CD15 (Lue-M1)

CK 5/6

mCEA

X

X

X

X

X

X


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EMA

Immunoreactivitypattern with EMA(epithelioid mesothelioma,pleural fluid).Mesothelioma cells withmembranous (arrow) andmembranous (arrow) andcytoplasmicimmunostaining. Note themicrovilli (arrowhead).[Immunostained cell-blocksection (100XZoomed)].

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a b HBME-1HBME-1

HBME-1 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid).Mesothelioma cells with membranous (arrow in a) and cytoplasmic immunostaining.Note the microvilli (arrowhead in b).


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Pan-cytokeratin


immunoreactivity pattern(pleural fluid).Reactive mesothelial cellswith cytoplasmicimmunostaining (arrow ininset). Some reactivemesothelial cells may showa concentrici t i i tt

Pan-cytokeratin

immunostaining patternaround the nucleus betterappreciated by adjustingfine focus.

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Cytokeratin 7 immunoreactivity pattern(epithelioid mesothelioma, pleural fluid). Neoplastic mesothelial

Cytokeratin 7

Neoplastic mesothelial cells with cytoplasmic immunostaining. Note the bushy microvilli (arrowhead).


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a b CalretininCalretinin

Calretinin immunoreactivity pattern (epithelioid mesothelioma, pleural fluid).Mesothelioma cells (arrow in a) show nuclear (arrowhead 1) immunoreactivity usually withcytoplasmic immunostaining (arrowhead 2) imparting the so called ‘fried-egg’ appearance.

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Calretinin immunoreactivity


RM

immunoreactivity pattern (pleural fluid).

Reactive mesothelial cells (blue arrows). The effusion also contains metastatic mammary

NC

Calretnin

ycarcinoma cells (red arrow NC).


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WT-1 immunoreactivity pattern


p(Metastatic colonic adenocarcinoma, peritoneal fluid).Reactive mesothelial cells(arrow RM) show nuclearimmunoreactivity (arrowheadin inset) with somecytoplasmic immunostaining.

RM

WT-1

y p gRare adenocarcinoma cellsdemonstrating nuclearimmunoreactivity for CDX2were also seen in othersection.

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B72.3 immunoreactivity pattern(Metastatic mammary adenocarcinoma, pleural fluid).Metastatic adenocarcinoma

NC

B72.3

Metastatic adenocarcinomacells (red arrow NC) show acytoplasmic immunoreactivitypattern.


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Vimentin


Vimentin immunoreactivity pattern (peritoneal wash).Reactive mesothelial cells(arrow RM) show cytoplasmicimmunoreactivity pattern(arrowhead in inset)

RM

vimentin

(arrowhead in inset).

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RM

LCA (CD45 ) immunoreactivity pattern (pleural fluid).Reactive mesothelial cells (blue arrow RM) with chronic inflammatory cells (redRM

LCA

inflammatory cells (red arrows). The inflammatory cells show a strong cytoplasmic immunoreactivity pattern obscuring the nucleus (arrowhead in inset).


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CD 68 (PGM1 ) immunoreactivity

tt (M i


H

H

H

pattern (Metastatic mammary adenocarcinoma with proliferation spheres (red arrow NC), pleural fluid).Histiocytes show CD68 immunoreactivity (blue arrows H). In our experience, PGM1 does not show non

NC

HCD68

PGM1 does not show non-specific immunostaining usually associated with KP1. Inset- Histiocytes (blue arrow H) with cytoplasmic immunoreactivity pattern around the nucleus.

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NC


NC

NC

a b BerEP4BerEP4

BerEP4 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid).a. The neoplastic cells in proliferation spheres (red arrow NC)- membranous immunostainingwith a honey comb-like pattern. b. Solitary adenocarcinoma cells (red arrow NC)- membranousimmunostaining pattern along the cell membrane (arrowhead in inset).


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NCNCComparison of immunoreactivity with


BerEP4a b BerEP4

NC

NC

NCNC

immunoreactivity with BerEP4 and B72.3(Metastatic mammary adenocarcinoma, pleural fluid).As Compared to B72.3, most of the adenocarcinoma cells (red arrows NC) show strong

c dB72.3 B72.3

NC) g

membranous BerEP4 immunoreactivity.

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Monoclonal CEA


m

(mCEA) immunoreactivity pattern(Metastatic ovarian carcinoma, peritoneal

fluid).Metastatic adenocarcinoma

mCEA

c

cells show cytoplasmic (c) and membranous (m) immunostaining.


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CDX2 immunoreactivity


NC

ypattern(Metastatic colonic adenocarcinoma,

peritoneal fluid).The adenocarcinoma cells show nuclear immunoreactivity (arrow

CDX2

NC). Compare with non-immunoreactive nuclei with blue hematoxylin counterstain (arrowhead).

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TTF-1 immunoreactivity


NC

ypattern(Metastatic pulmonary carcinoma, pleural fluid).The solitary adenocarcinoma cells as the predominant population (arrows NC) show nuclear immunoreactivity

TTF-1NC

(arrowheads in inset).


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mMOC-31 immunoreactivity pattern(Metastatic mammary carcinoma, pleural fluid). The adenocarcinoma cells show predominantly

MOC-31

c

membranous (m) with cytoplasmic (c) immunoreactivity.

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Diffuse malignant mesothelioma of epithelial


EM

A

a b

mesothelioma of epithelial type, (pleural fluid).Neoplastic cells are immunoreactive for EMA (a & b) and HBME-1 (c, d, & e) with a membranous immunostaining pattern (arrows) highlighting long, slender, microvilli (arrowheads).

c d e

HB

ME

-1


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a bEMA HBME-1

Adenocarcinoma, peritoneal fluid.Cytoplasmic immunostaining pattern (arrows) with focal blotchy immunostaining for EMA (a) and HBME-1 (b) along the cell membrane .

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For reproducible results, it is important to select any


immunopanel which will fundamentallyidentify most of the mesothelial and inflammatory cellsto create the basic mapfor confirmation of a ‘second-foreign’ population by ‘SubtractiveCoordinate Immunoreactivity Pattern’ (SCIP) approach

* Shidham VB, Atkinson BF. Immunocytochemistry of effusion fluids: Introduction to the SCIP approach. In: Shidham VB and Atkinson BF. Editors ‘Cytopathologic Diagnosis of Serous Fluids’ First edition, Elsevier (W. B. Saunders Company); 2007. Ch 5, pp. 55-78.

*Shidham VB. Diagnostic Cytopathology of Serous Fluids- A brief review of cytomorphologic and SCIP approach. CytoJournal (In press).


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Evaluation of‘Subtractive Coordinate Immunoreactivity Pattern’

(SCIP)

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Met

asta

sis

(no

n-c

arci

no

ma)

2

3

6

57

2

3

1

6

57

2

3

1

6

57

3

1

6

57

2

3

1

6

57

ZImmunocytochemistry of

effusion fluids

2

6 8

ial &

ry

cel

ls

2

6 8

2

6 8

2

6 8

2

6 8

X

1 54 1 54 1 54 1 541 54

Met

asta

sis

(car

cin

om

a)

21

54

3

7

6

21

54

3

7

6

21

54

3

7

6

21

54

3

7

6

21

54

3

7

6

Y

SCIP approach

3

1

5

4 7

Mes

oth

elin

flam

mat

or

3

1

5

4 7

3

1

5

4 7

3

1

5

4 7

3

1

5

4 7

vim

en

tin

Pan

CK

(Mix

ture

of A

E1/

AE

3 &

CA

M5

.2)

Cal

reti

nin

WT

-1

LC

A(C

D45

)[o

r P

GM

1(C

D68

) o

r m

ixtu

re o

f LC

A

& P

GM

1]

A B C D E


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SCIP approachapproach

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SCIP approach


a b


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CIP lret

inin

, D2

-40

)

Carcinoma

Immunoreactive for ‘negative’ mesothelial markers such as-BerEP4, B72.3, MOC-31, mCEA, .

Proceed with:Immunopanel for unknown primary OR

CK+,vim –/+


pa

ne

l fo

r e

valu

ati

on

by

SC

K7,

CK

20, B

er/E

P4,

B72

.3, M

OC

31C

al

Without‘second foreign’ population

With‘second-foreign’ population

Qualitative & quantitative features of mesothelioma

CK-,vim+

Lymphoma

Melanoma/Sarcoma

LCA+

LCA–S-100 protein &

Melanoma markers

Lymphoma panelCytogenetics

Gene rearrangement

Restricted panel for known primary

+–

Ba

sic

p(v

imen

tin

, Pan

CK

,CK second-foreign population features of mesothelioma

Negative for malignancy

Absent Present

Sa

rco

ma

Me

lan

om

a

Immunopanel for sarcomaORRestricted panel for known primary

Malignantmesothelioma

EMA/HBME-1: Microvillous pattern

B72.3–, BerEP4–

37


Short course


SCIP approach

B. Pan-cytokeratinImmunoreactive

A. VimentinNon-immunoreactive

‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections

NC

10X 40X

NC

NC

NCapproach

C. LCA (CD45)Non-immunoreactive

D. CalretininNon-immunoreactive(Inset {2}-Mesothelial cell immunoreactivenuclear-cytoplasmic)

RM

10X

10X

10X 40X

40X

40X

NC

NC

NC

NC

NCRM

Metastatic colonic adenocarcinoma, (peritoneal fluid).

F. CDX2Immunoreactivenuclear

HEstained cell blocksection

y p )

E. WT-1Non-immunoreactive(Arrow 2 with inset: Mesothelial cell-immunoreactivenuclear-cytoplasmic)

40X

RM

10X

10X

10X

40X

40X

100X40X

NC

NC

NC

NC

RM

RM

NC


Short course


B. Pan-cytokeratinImmunoreactive



10X

NC

RM

Zoomed

SCIP approach


C. CalretininNon-immunoreactive[Mesothelial cells (RM) immunoreactivenuclear-cytoplasmic]

D. BerEP4Immunoreactive

10X

10X

10X

Zoomed

NC

RM

Zoomed

NCRM

approach

sect o

E. Cytokeratin 7Immunoreactive

F. Cytokeratin 20Non-immunoreactive

10X

10X

10X

10X

Zoomed

Zoomed

NC

RM

Metastatic ovarian carcinoma, (peritoneal fluid).

38


Short course


B. CD68 (PGM1)



40X

SCIP approach

Metastatic mammary adenocarcinoma, (pleural effusion).

Non-immunoreactive

C. CalretininNon-immunoreactiveMesothelial cell (RM)immunoreactivenuclear-cytoplasmic)

40X

40X

RM

RM

approach


D. Cytokeratin 7Immunoreactive

E. BerEP4Immunoreactive

40X40X

40X

NC

NC

RM

NC

RM


Short course


B. CD68 (PGM1)Non-immunoreactive

A. VimentinNon-immunoreactive(Mesothelial & inflammatory cells are immunoreactive)


20X 40X

Metastatic mammary adenocarcinoma,

SCIP approach

C. CalretininNon-immunoreactive(Rare mesothelial cell [blue arrow] is immunoreactivenuclear-cytoplasmic)

(inflammatory cells are immunoreactive)

20X

20X

40X

40X

RM

mammary adenocarcinoma, (pleural effusion).

approach

D. BerEP4Immunoreactive

E. Estrogen receptorsImmunoreactive

20X

20X

40X

40X

NC

NC

NC

39


Short course


B. Cytokeratin 20Non-immunoreactive

A. Cytokeratin 7Immunoreactivecytoplasmic

40X 100X


NC

SCIP approach

C. TTF-1Immunoreactive Nuclear

D. ChromograninImmunoreactivecytoplasmic

40X 100X

40X 100X

NC

NC Metastatic small cell carcinoma,(pleural fluid).

approach

F. CD56Immunoreactivecytoplasmic

E. SynaptophysinWeak immunoreactivecytoplasmic

40X 100X

40X 100X

100X40X

NC


Short course


‘Subtractive coordinate immunoreactivity Pattern’ (SCIP) in cell block sections

A. Cytokeratin 7Non-immunoreactive[Mesothelial cell Immunoreactive (red arrow)Cytoplasmic]

B. CalretininNon-immunoreactive[M th li l ll

PAP stained Cytospin preparation (a-c)

40X

RM

RM

SCIP approach[Mesothelial cell

Immunoreactive (red arrow)nuclear-cytoplasmic]

C. CD 20ImmunoreactiveCytoplasmic (red arrow)

a

b

10X

40X

40X

40X

NC

RM

NC

Large B-cell lymphoma, (peritoneal fluid).

approach(continued)

D. Bcl2ImmunoreactiveCytoplasmic (red arrow)

E. CD3Non-immunoreactive

HE stained cell blockSection (d)

c

d

NC

NC

40X 40X

40X

NC

NC

40


Short course


SCIP with dual

Vimentin (Brown)-Cytokeratin 7 (Red)Vimentin (Brown)-Cytokeratin 7 (Red)


dual staining method

a bVimentin (Brown)-Cytokeratin 7 (Red)Vimentin (Brown)-Cytokeratin 7 (Red)

Mammary carcinoma, (effusion fluid).

dc

Shidham V.B., Varsegi G, D’Amore K.Two-color immunocytochemistry forevaluation of effusion fluids for metastaticadenocarcinoma.CytoJournal 2010 Feb 10;7:1


Short course


SCIP with dual staining method

C l ti i (B ) B EP4 (R d)Vi ti (B ) C t k ti 7 (R d)

Metastatic mammary adenocarcinoma, pleural fluid.


Calretinin (Brown)-BerEP4 (Red)Vimentin (Brown)-Cytokeratin 7 (Red)

a b c

41


Short course


SCIP with dual staining methodMetastatic gastric adenocarcinoma, peritoneal fluid.


Calretinin (Brown)-BerEP4 (Red)Vimentin (Brown)-Cytokeratin 7 (Rred)

a b c


Short course


Evaluation of unknown primary sites of origin- Where do they come from?


on

cyt

olo

gy

1 Equivocal for

malignant cells

2

Negativefor

malignant cells

3

Eff

usi

o

Unequivocal for

malignant cells

4

malignant cells

42


Short course


N t il bl 9

Suspicious for malignant cells with recommendation to

13

Evaluation of unknown primary sites of origin- Where do they come from? (continued)

Evaluation of unknown primary sites of origin

Cell-blockNegative for malignant cells

Report17

Immunocytochemical

Not availableOr insufficient

9submit additional specimen forconfirmation with additional cytopathological evaluation withcell-block preparation if effusion reaccumilatesb.

Report

13

Eq

uiv

oca

l fo

r al

ign

ant

cell

s

2

5

Positive for malignant cells,depending on results of immunocytochemistry,comment about the primary site

Report

18

for malignant cellscharacterization on cell-block sections to confirm presence of second population by SCIP with characterization of this second population for possible primary site

12

Available8

m


Short course




Evaluation of Primary

Negativefor

malignant cells

3Negative for malignant cells

Report6

43


Short course





Clinical correlationc

7

Possible10 Comparative

review of primary lesion

14

neq

uiv

oca

l fo

rli

gn

ant

cell

s

4

Not possible11

Un

ma


Short course


Cytomorphological patterns Possible primary carcinoma

Cytomorphological features suggestive of a primary site


y p g p p y

1 Three dimensional round cell groups-proliferation spheres or ‘cannonballs’

Breast adenocarcinomaOvarian adenocarcinomaMesothelioma of epithelioid typeReactive mesothelial proliferations

2 Acini / glands Adenocarcinomas of Breast, Lung,Colorectum, Stomach, Ovary,Endometrium, etc.Mesothelioma of epithelioid typeMesothelioma of epithelioid type

3 Predominantly scattered isolated malignant cells

Gastric adenocarcinomaNon-cohesive variant of lungadenocarcinomaBreast lobular carcinomaAdrenocortical carcinoma(Also Lymphoma, Melanoma, & Sarcoma)

44


Short course



Cytomorphological features suggestive of a primary site (continued)


4 Carcinoma cells in chains and rows (‘Indian file’ pattern)

Breast- Lobular and ductal carcinomaPoorly differentiated small cell carcinomaGastric adenocarcinomaOvarian adenocarcinoma

5 Extensive cytoplasmic vacuolization Renal cell adenocarcinoma (glycogen, fat)Adrenocortical carcinoma (fat)Benign mesothelial cellsPancreatic adenocarcinoma (mucin)( )Ovarian adenocarcinoma (mucin)Lung adenocarcinomaClear cell carcinoma endometrium

6 Signet ring cells Gastric adenocarcinomaColorectal adenocarcinoma

7 Intracytoptasmic lumina Breast adenocarcinoma


Short course





y p g p p y

8 Giant tumor cells Lung large cell carcinoma- giant cell typePancreatic adenocarcinomaThyroid anaplastic carcinomaSquamous cell carcinoma(Also Melanoma & pleomorphic sarcoma)

9 Targetoid intracytoplasmic vacuole containing secretion

Breast adenocarcinoma (especiallylobular)Thyroid carcinoma (colloid)Thyroid carcinoma (colloid)Ovarian carcinomaPancreatic carcinoma

10 Three dimensional groups in papillary configurations

Bronchioloalveolar carcinomaColonic adenocarcinomaEndometrial adenocarcinomaMammary adenocarcinoma

45


Short course



11 Three dimensional papillary groups Ovarian carcinoma- serous papillary



11 Three dimensional papillary groups containing psammoma bodies

Ovarian carcinoma- serous papillaryThyroid papillary carcinomaPancreatic papillary carcinoma

12 Cell groups of tall columnar cells with a picket fence pattern

Colonic adenocarcinomaPancreato-biliary carcinoma

13 Cellular pleomorphism Poorly differentiated carcinomas of lung,pancreas, ovary, thyroid, urothelium

14 Large polyhedral cells Hepatocellular carcinomag p y pTransitional cell carcinomaLarge c ell type squamous cell carcinoma

15 Cytoplasmic pigment Hepatocellular carcinoma- Bile,(Melanoma- melanin)

16 Prominent nucleoli Hepatocellular carcinomaRenal cell carcinomaProstatic adenocarcinoma


Short course


Cytomorphology

Evaluation of unknown primary sites of origin-Where do they come from? (continued)

Positive f li t ll

Report

19



Clinical correlationc

7

Possible10

Cytomorphologynot classical for the known primary neoplasm 16

Cytomorphologyconsistent withprimary site 15Comparative

review of primary lesion

14

Positive for malignant cells with broad

Report26

neq

uiv

oca

l fo

rli

gn

ant

cell

s

4

for malignant cells,consistent withmetastatic cancer from previous neoplasm.

Not possible11

Cell-block20

Available 21

Not availableOr insufficient

22

Immunocytochemical characterization

23

cells with broad cytomorphological characterization (such asnon-small cell carcinoma vs small cell carcinoma vs lymphoma). Recommend additional specimen for cell-blockfor immunocharacterization of neoplastic cells if effusion reaccumilates.

Un

ma

46


Short course



Report

27



Immunoprofile of the 25

Immunoprofile of the second population isconsistent with primary neoplasm.

24

no

cyto

chem

ical

ar

acte

riza

tio

n

23Positive for malignant cells,consistent with XYZ primary.

28Immunoprofile of the second population is not distinct for primary neoplasm.

25

Imm

un

cha

Positive for malignant cells,And suggest a differential diagnosis for the primary sites.

Report


Short course


CytoJournalwww cytojournal com

End

www.cytojournal.com

[email protected] presentation is available on web for all conference attendees at- http://alturl.com/3ucwx

ser fl cytol ico pune jan 7, 2012 (handout)

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