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

Diagnostic Cytopathology
International CME on Hotel O, Pune, India of Effusion Fluids
Oncopathology January 7, 2012 Short course

of Effusion Fluids
Short course

Vinod B. Shidham, MD, FRCPath, FIAC
Vice-chair & Professor
Director of Cytopathology, Cytotechnology School, Cytopathology fellowship
Dept of Pathology, Wayne State University Medical School
Detroit, MI 48201, USA
Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com)
vshidham@med.wayne.edu

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

International CME on of Effusion Fluids
Oncopathology Short course

Disclosure
I 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
Co-author of th chapter**
C th f the h t
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.

1

Oncopathology Acknowledgment Short course

Many images and tables are based on chapters in:

Shidham VB and Atkinson BF.
Cytopathologic Diagnosis of Serous
Fluids
Elsevier (W. B. Saunders Company) First
edition, 2007.


Outline

Part 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?

2


Anatomy, histology, cytology, and effusions

a. Peritoneal cavity
y
b. Left & right pleural cavities
c. Pericardial cavity

Four major serous cavities


Anatomy, histology, cytology, and effusions (continued)
1
Histology of serous li i
Hi t l f lining
(inguinal hernia sac). The
mesothelial cells lining the
fibrous tissue are flat (1).
Focal reactive changes are
seen as hypertrophy of
a b some cells which assume a
2 3 cuboidal contour (2,3).
( ,3)
[a-d, HE stain (a, 10X; b-d,
100X)].

c d

3



Peripheral light ectoplasm (1)
Mesothelial cells (peritoneal
fluid): show outer faintly stained
Inner darker endoplasm (2)
ectoplasm (1) with inner denser
endoplasm (2) rich in
Slightly off-center nucleus intermediate filaments. The
nucleus is usually central or near
Nucleolus
central (a), but may be eccentric
(b). Nucleoli are readily
1 1 observed.
observed The vacuolation
generally begins at the periphery
in ectoplasm (1).
2 [a,b, Papanicolaou stained
2
SurePath® Preparation (a,b,
100XZoomed)]

a b



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 inner
endoplasm. The nucleus is central to slightly eccentric but not touching the cell
periphery. The cell margin shows blebs and is ruffled.
[Diff-Quik stained Cytospin preparation (100XZoomed)]

4


Bloody effusions
(Hemothorax,
(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
Para pneumonic effusions Pancreatitis
Post traumatic effusions Acute aortic dissection

underlying malignancy
-Post cardiothoracic procedures / surgeries
-Thoracic cavity vascular damage
Pulmonary embolism
Endometriosis
Sarcoidosis
Intralobar pulmonary sequestration
Asbestos exposure associated pleural effusion Some Infections –e.g B. Anthrax


Collection, transportation, and processing
Biopsy vs effusion cytology

5


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
[e g di sodium EDTA dihydrate (EDTA Na2, 2H2O) 1 4 mg per ml of effusion fluid]
1.4

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 specimen
may be achieved by density gradient centrifugation with Ficoll

International CME on Collection, transportation, Diagnostic Cytopathology
of Effusion Fluids
Oncopathology and processing (continued) Short course

Collect fresh effusion fluid Concentrate the
with or without anticoagulant effusion fluid specimen

Concentrate the
remaining fluid similar to
Direct smear of Specimen specimen without clot
unconcentrated specimen for with clot
semi-quantitative evaluation Use homogenized specimen after mixing
(For paucicelluar fluids: Use cell-rich sediment)

Concentrate by centrifugation
Remove the clot (at 600g for 10 minutes)
and process for
cell-block
preparation Pour off supernatant and
vortex to resuspend cell pellet
Process for paraffin-
embedding after formalin Cell-block preparation Smear preparation
fixation. Histogel • Direct smears • SurePrep (Autocyte)
Plasma-Thrombin • Cytospin • ThinPrep
Celloidin bag • Other methods

6



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 stain
ii. 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)
ii. Air-dried-
ii Ai d i d
Diff-Quik stain
Pap stain: After rehydration in saline and
post-fixation in 95%ethanol with 5% acetic acid (28)
c. Filters
d. Liquid based cytology (SurePathTM or ThinPrepTM or other non-proprietary methods)



Preparation Purpose
Semiquantitative evaluation of
A Diff-Quik stained direct smear
of the undiluted specimen cellularity.
Diff-Quik stained air-dried Rapid initial detection of second
B Shandon EZ Megafunnel™ population and cytomorphologic
preparation evaluation of hematopoietic
cells.
Pap stained preparation Cytomorphologic evaluation
C especially nuclear details.
details

Cell-block preparation in Evaluation of-
D HistoGelTM a. Immunoprofile,
b. Other special stains,
c. Architecture,

7


Approach to diagnostic cytopathology of effusions

► GENERAL FEATURES

► PROCESSING RELATED

► INTERPRETATION STRATEGY

► CYTOMORPHOLOGY

► IMMUNOCYTOCHEMISTRY


Approach to diagnostic cytopathology of effusions (continued)

► GENERAL FEATURES
►A general pathology l b
Any l h l laboratory may receive effusion- all general pathologists and
i ff i ll l h l i d
cytopathologists should be conversant with the diagnostic challenges and pitfalls
of effusion fluid cytology.
►Finding neoplastic cells in effusion specimens not only reveals that a patient has
cancer, but it also denotes the advanced nature of the disease which at this stage is
almost always incurable.
►The morphologic features of most of the cancer cells in effusion smears are
different f
diff t from th
those seen i exfoliative, b hi
in f li ti brushing, and FNA cytology.
d t l
►The standard cytologic criteria of malignancy based on evaluation of single cell
morphology 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 native
shapes of cancer cells cannot be a guiding feature.

8



► 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.



► INTERPRETATION STRATEGY
a. Reactive mesothelial cells- morphologic overlap with neoplastic cells
b. A single population- favor mesothelial cells
c. Second foreign population consistent with metastatic neoplasm
d. Sarcomas- previous history known
e. Romanowsky stains highlight the ‘second population’
f. Immunocytochemistry- objective confirmation of ‘second population’
y y
g. Identical orientation of serial sections for immunocytochemistry
h. Quantitative and qualitative clues for mesothelioma
i. Apoptosis

9



► CYTOMORPHOLOGY
1. Cell groups and intercellular cohesion
Non-cohesive, good intercellular cohesion, proliferation spheres

2. Arrangement of neoplastic cells
Papillary configurations

3. Cytoplasm of neoplastic cells

4. Special structures and cytological features

5. Other features

International CME on Algorithm for evaluation of Diagnostic Cytopathology
of Effusion Fluids
Oncopathology ‘second foreign population’ Short course

Reactive- 4
Usually single cells without large 3-D
groups
Mesothelial cells 2
Neoplastic- Mesothelioma 5
♦Quantity- Many cells
♦Quality- Many large groups
Cells in effusion fluid 1 Reactive- 6a
Hematopoietic cells 6 Inflammatory cells
(Non-cohesive cells)
Neoplastic- 6b
Non mesothelial cells 3
Non-mesothelial Lymphoma
L h
7
Neoplastic-8 (2nd foreign population) 8 ¶Metastatic cancer cells may be the predominant
Carcinoma (Cohesive cells) cells without being seen as a ‘second population’.
Sarcoma They may be present as scattered solitary cells with
(Spindle cells may be present. Known cytomorphology overlapping with floridly reactive
history of sarcoma is usually crucial for mesothelial cells. If indicated, immunocytochemistry
proper interpretation) would facilitate confirmation of these cells as non-
Melanoma (Non-cohesive cells) mesothelial.

10


The panorama of different faces of mesothelial cells
REACTIVE MESOTHELIAL CELLS
Binucleation and multinucleation-
multinucleation
Gigantic nuclei
Phagocytic activity
Cohesive Clusters and/or Papillary Structures
‘The two cell population approach’
Cell-in-cell configuration
Cells in sheets
Groups 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


The panorama of different face of mesothelial cells (continued)

a b c
Mesothelial 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)].

11



Panorama of mesothelial cells
(asc t c u d) Ce t a
(ascitic fluid). Central to near
ea
central nuclei. Rare mesothelial
a b c d e f cells may show eccentric nuclei
touching the cell membrane,
but usually there is a narrow
rim of cytoplasm separating the
g h i j k l nucleus from the cell border
(arrows).
[a-x, Diff Q ik stained cytospin
[ Diff-Quik t i d t i
preparations (a-x, 100x
m n o p q r
Zoomed)].

s t u v w x



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)].

12



Mesothelial cells with central to
eccentric nuclei (
ti l i (ascitic fl id)
iti fluid).
a b c d e f
Spectrum of cytomorphological
features.
[a-zc, Papanicolaou stained
g h i j k ThinPrep preparation (a-zc, 100x
Zoomed)]

l m n o p q

r s t u v w

x y z za zb zc



Mesothelial cells with
eccentric nuclei (ascitic
fluid). Careful scrutiny
usuallyy shows a
narrow rim of
cytoplasm separating
the nucleus from the
cell border (arrows).
[Papanicolaou stained
ThinPrep preparation,
l x 100XZoomed].

13



NC NC
RM
IC
RM IC

DQ Pap

a b
Metastatic adenocarcinoma (pleural fluid). An example with morphologically identifiable unequivocal
‘second foreign population’ (red arrow NC) other than mesothelial cells (blue arrow RM) and inflammatory
cells (brown arrow ‘IC) in DQ and PAP stained preparations. This ‘second population’ of cells ’ (red
arrow 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, 100x
Zoomed)]



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

14


Factors leading to potential diagnostic pitfalls
a. Surface tension related alterations in cytomorphology
b.
b Improper specimen processing
c. Many faces of reactive mesothelial cells
d. Proliferation related features
i) Proliferation spheres
ii) Increased number of mitotic figures
iii) Prominent nucleoli
e. Degenerative changes
Nuclear hyperchromasia
Cytoplasmic vacuolation
C l i l i
f. Presence of some unexpected patterns and unusual structures
i) Reactive lymphoid population
ii) Polymorphic lymphocytes
iii) Single population of cells due to predominance of tumor cells
iv) Psammoma bodies
v) Three dimensional benign cell groups
Benign papillary inclusions, Gland-like epithelial structures, Mullerian inclusions
vi) Megakaryocytes


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. Lack
c L k off exfoliation b llow-grade sarcomas and spindle cell mesotheliomas.
f li ti by d d i dl ll th li

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

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

15


Part II


Immunocytochemistry of effusion fluids

The most important issue to be considered when applying
immunocytochemistry to effusion fluids is the significant
variation in results due to the many variables incurred
from the time of collection of the specimen to its final
immunostaining.

16

International CME on Immunocytochemistry of of Effusion Fluids
Oncopathology effusion fluids Short course

UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY

Confirmation of a ‘
C fi ti f ‘second-foreign’ non-inflammatory population of cells other th
df i ’ i fl t l ti f ll th than
mesothelial cells in effusions correlate with metastatic cancer with objectivity.

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

It is crucial to:
►Orient the serial sections identically on all slides
(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.


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-dried
smears rehydrated and post-fixed in formol alcohol, liquid based cytology
preparations- SurePath or ThinPrep, cytospin preparations, etc) should be
avoided.

For reproducible results a standardized protocol with steps
comparable to the processing of formalin-fixed paraffin-embedded
tissue sections is essential.

17


Critical issues leading to
the best result with cell blocks
of cytology specimens
with singly scattered cells
Aligning the cells along the cutting surface

Monitor the depth of section cutting


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

18


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


From:
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160

19


Modified from:
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160


From:
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160

20


The video article is available FREE on the web
under open access at:
http://www.jove.com/index/Details.stp?ID=1316
Video f J VE ti l
Vid of JoVE article (8 minutes 15 sec)
i t )

Video of JoVE article (8 minutes 15 sec)


Immunophenotyping and cell blocks-
Factors affecting immunoreactivity-
Loss, reduction, or enhancement of antigen immunoreactivity
Exposure to different reagents and fixative(s)
Temperature
Storage of specimen with or without fixative

Subtractive Coordinate Immunoreactivity Pattern (SCIP) approach
Shidham & Atkinson
Ch 5 Imm noc tochemistr of effusion fl uids: introd ction to SCIP approach
5. Immunocytochemistry eff sion ids introduction 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
approach
CytoJournal (In press).

21


Discrepant results between formalin-fixed paraffin-embedded tissue sections of
surgical pathology material and effusion fluid cell block sections are not uncommon
cell-block 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
(i e heat induced epitope retrieval enzyme digestion etc )
retrieval, digestion, etc.),
antibody clones used,
antibody titer, and
other variations in immunostaining protocols.
The proteinaceous effusion fluid around suspended cells may contribute to
unexpected nonspecific immunoreactivity.


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 of
effusion fluid collected.

To avoid inadequate resubmission, it may be specifically communicated in the
recommendation as comment :
“Recommend submission of most of the drained effusion fluid (up to
1000 mL). Larger volume of specimen facilitates retrieval of adequate
cellular material in cell-block sections for immunocytochemical
evaluation”.

22


Interpretation:
I t t ti
All aspects of individual and complimentary immunomarkers
should be considered collectively rather than applying a
reflexive positive-negative approach.

Evaluation of co-ordinate immunoreactivity in different cell
y
population.


Immunostaining None Nuclear Nuclear & Cytoplasmic Membranous Microvillous
pattern cytoplasmic

Immunomarker

Calretinin X X

WT-1 X X

D2-40 (Podoplanin) X

Cytokeratin* X

Vimentin X

LCA (CD45) X

PGM1 (CD68) X

EMA X AdCa X meso

HBME-1 X AdCa X meso

23


Immunostaining None Nuclear Nuclear & Cytoplasmic Membranous Microvillous
pattern cytoplasmic

Immunomarker

B72.3 X

BerEP4 X

Cadherins X

MOC-31 X

CD44S X

Mesothelin X

mCEA X

CK 5/6 X

CD15 (Lue-M1) X

CA19.9 X

Ttf-1 X


Immunoreactivity
pattern with EMA
(epithelioid mesothelioma,
pleural fluid).
EMA Mesothelioma cells with
membranous (arrow) and
cytoplasmic
immunostaining. Note the
microvilli (arrowhead).
[Immunostained cell-block
section (100XZoomed)].

24


a HBME-1 b HBME-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).


Pan-cytokeratin
immunoreactivity pattern
(pleural fluid).
Reactive mesothelial cells
with cytoplasmic
immunostaining (arrow in
inset). Some reactive
mesothelial cells may show
a concentric
immunostaining
i t i i pattern
tt
around the nucleus better
appreciated by adjusting
fine focus.

Pan-cytokeratin

25


Cytokeratin 7
immunoreactivity
pattern
(epithelioid mesothelioma,
pleural fluid).
Neoplastic mesothelial
cells with cytoplasmic
immunostaining. Note the
bushy microvilli
(arrowhead).
Cytokeratin 7


2
1

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

26


Calretinin
immunoreactivity
pattern (pleural fluid).
Reactive mesothelial
cells (blue arrows).
The effusion also
contains metastatic
RM
mammary y
carcinoma cells (red
arrow NC).

NC
Calretnin


WT-1 immunoreactivity
p
pattern
(Metastatic colonic
adenocarcinoma,
peritoneal fluid).
(arrow RM) show nuclear
immunoreactivity (arrowhead
in inset) with some
cytoplasmic immunostaining.
y p g
Rare adenocarcinoma cells
demonstrating nuclear
immunoreactivity for CDX2
RM were also seen in other
section.
WT-1

27


B72.3
immunoreactivity
pattern
(Metastatic mammary
adenocarcinoma, pleural
fluid).
Metastatic adenocarcinoma
cells (red arrow NC) show a
cytoplasmic immunoreactivity
pattern.
NC

B72.3


Vimentin
immunoreactivity
pattern
(peritoneal wash).
(arrow RM) show cytoplasmic
immunoreactivity pattern
(arrowhead in inset)
inset).
RM

vimentin

28


LCA (CD45 )
immunoreactivity
pattern
(pleural fluid).
(blue arrow RM) with chronic
RM inflammatory cells (red
arrows). The inflammatory
cells show a strong
cytoplasmic immunoreactivity
pattern obscuring the nucleus
(arrowhead in inset).
LCA


CD 68 (PGM1 )
immunoreactivity
pattern (M
tt (Metastatic
i
mammary adenocarcinoma
H with proliferation spheres
H (red arrow NC), pleural
fluid).
Histiocytes show CD68
H
immunoreactivity (blue
arrows H). In our experience,
PGM1 does not show non non-
specific immunostaining
usually associated with KP1.
NC Inset- Histiocytes (blue arrow
H) with cytoplasmic
H immunoreactivity pattern
CD68 around the nucleus.

29


NC
NC

NC
a BerEP4 b BerEP4

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


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

c B72.3 d B72.3

30


Monoclonal CEA
(mCEA)
m immunoreactivity
pattern
(Metastatic ovarian
carcinoma, peritoneal
fluid).
Metastatic adenocarcinoma
cells show cytoplasmic (c)
and membranous (m)
c immunostaining.

mCEA


CDX2
y
immunoreactivity
pattern
NC
(Metastatic colonic
adenocarcinoma,
peritoneal fluid).
The adenocarcinoma cells
show nuclear
immunoreactivity (arrow
NC). Compare with non-
immunoreactive nuclei with
blue hematoxylin
counterstain (arrowhead).

CDX2

31


TTF-1 immunoreactivity
y
pattern
(Metastatic pulmonary
carcinoma, pleural fluid).
The solitary adenocarcinoma
cells as the predominant
population (arrows NC) show
NC nuclear immunoreactivity
(arrowheads in inset).

NC
TTF-1


MOC-31 immunoreactivity
m pattern
(Metastatic mammary
carcinoma, pleural fluid).
The adenocarcinoma cells
show predominantly
membranous (m) with
cytoplasmic (c)
immunoreactivity.
c
MOC-31

32


Diffuse malignant
mesothelioma of epithelial
type,
EMA

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

(arrowheads).

c d e


a EMA b HBME-1

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

33


For reproducible results, it is important to select any
immunopanel which will fundamentally
identify most of the mesothelial and inflammatory cells
to create the basic map
for confirmation of a ‘second-foreign’ population by ‘Subtractive
Coordinate 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).


Evaluation of
‘Subtractive Coordinate Immunoreactivity Pattern’
(SCIP)

34

Mesothelial & X Metastasis Y Metastasis Z
ry
inflammator cells (carcinoma) (non-carcinoma)

vimentin 1 2 1 2 1 2
A
3 3
4 3 4
6 5 6
5 4

7 6
8 7 5
7

SCIP
1 2 1 2 2
B Pan CK 1

approach
(Mixture of AE1/AE3 3
3
& CAM5.2) 4 3
6 5 4 6
4

Oncopathology
Oncopathology

5
7 6

International CME on
International CME on

8 7 5
7

1 2 1 2 2
C LCA(CD45) 1
[or PGM1(CD68)
3
3
or mixture of LCA 4 3
6 5 4 6
& PGM1] 4
5
7 6
8 7 5
7

1 2 1 2 2
D Calretinin 1
3
3
4 3
6 5 4 6
5 4

effusion fluids
effusion fluids

7 6
8 7 5
7

Immunocytochemistry of
Immunocytochemistry of

1 2 1 2
E 1
WT-1 3 3
4 3
6 5 4 6
5 4

7 6
8 7 5
7
SCIP

Short course
Short course

of Effusion Fluids
of Effusion Fluids

approach


35


SCIP
approach

a b

International CME on Immunocytochemistry of
of Effusion Fluids
Oncopathology effusion fluids
Short course

Immunoreactive for ‘negative’
lretinin, D2-40)

mesothelial markers such as-
BerEP4, B72.3, MOC-31, mCEA, .
Proceed with:
CK+,vim –/+ Carcinoma Immunopanel for unknown primary
CIP

OR
Basic panel for evaluation by SC
(vimentin, PanCK,CK CK20, Ber/EP4, B72.3, MOC31Cal

Restricted panel for known primary
With
‘second-foreign’ population LCA+
Lymphoma panel
Lymphoma Cytogenetics
Gene rearrangement
CK-,vim+
Melanoma/ S-100 protein &
LCA– Sarcoma Melanoma markers

Without Qualitative & quantitative – +
K7,

‘second foreign’ population features of mesothelioma
p

second-foreign
Melanoma
Sarcoma

Absent Present

Negative for Malignant
malignancy mesothelioma
EMA/HBME-1: Immunopanel for sarcoma
Microvillous pattern OR
B72.3–, BerEP4– Restricted panel for known primary

36

‘Subtractive coordinate immunoreactivity pattern’
(SCIP) in cell block sections
Oncopathology A. Vimentin Short course
Non-immunoreactive NC
NC
10X 40X

SCIP B. Pan-cytokeratin
Immunoreactive

approach NC

10X
NC
40X

C. LCA (CD45)
Non-immunoreactive NC
Metastatic colonic
NC
10X 40X adenocarcinoma,
D. Calretinin
Non-immunoreactive
(peritoneal fluid).
(Inset {2}- RM
Mesothelial cell NC
immunoreactive
nuclear-cytoplasmic) RM
y p ) NC
10X 40X

E. WT-1
Non-immunoreactive RM
HE (Arrow 2 with inset:
stained Mesothelial cell- NC
cell block immunoreactive RM NC
section nuclear-cytoplasmic) 10X 40X

NC
F. CDX2
Immunoreactive
nuclear NC
NC
40X 10X 40X 100X

International CME on RM
of Effusion Fluids
Oncopathology A. Vimentin Short course
Non-immunoreactive
NC
10X Zoomed

SCIP B. Pan-cytokeratin
Immunoreactive

approach 10X

RM
C. Calretinin NC
Non-immunoreactive
[Mesothelial cells (RM)
immunoreactive
nuclear-cytoplasmic]
10X Zoomed

RM

HE D. BerEP4
stained Immunoreactive NC
cell block
sect o
section 10X Zoomed

E. Cytokeratin 7
Immunoreactive
Metastatic ovarian carcinoma,
(peritoneal fluid).
10X 10X
NC

RM F. Cytokeratin 20
Non-immunoreactive

Zoomed 10X

37

A. Vimentin
Non-immunoreactive

40X

SCIP
approach B. CD68 (PGM1)
Non-immunoreactive

40X

RM
C. Calretinin
Non-immunoreactive
Mesothelial cell (RM)
immunoreactive Metastatic mammary
nuclear-cytoplasmic)
40X
adenocarcinoma,
(pleural effusion).
RM

D. Cytokeratin 7
HE
Immunoreactive
stained
cell block NC
section
40X

RM
NC

E. BerEP4
Immunoreactive
NC
40X 40X

Oncopathology
A. Vimentin Short course
Non-immunoreactive
(Mesothelial &
inflammatory cells are
immunoreactive)
20X 40X

SCIP Metastatic
B. CD68 (PGM1)
approach Non-immunoreactive
(inflammatory cells are
mammary adenocarcinoma,
immunoreactive) (pleural effusion).
20X 40X

RM
C. Calretinin
Non-immunoreactive
(Rare mesothelial
cell [blue arrow] is
immunoreactive
nuclear-cytoplasmic)
20X 40X

D. BerEP4
Immunoreactive NC

20X 40X

NC

E. Estrogen
receptors
NC
Immunoreactive

20X 40X

38

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

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

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