cell block on
TRANSCRIPT
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
May 17 & 19, 2010Vinod B. Shidham, MD, FRCPath, FIAC
Professor
Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com)
Vice-chair
Director of Cytopathology, Cytotechnology School, Cytopathology fellowship, &
GI fellowship
Dept of Pathology, Wayne State University Medical School
Detroit, MI 48201, [email protected]
Cell Blocks
In Cytopathology(Workshop# 5 & 43)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Disclaimer
VS is co-editor of
‘Cytopathologic Diagnosis of
Serous Fluids’Elsevier (W. B. Saunders Company)
cited for various methods of
cell block preparation (the
sketches and tables used are
from this reference).
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Disclaimer
VS has indirect financial interest (through spouse) in
AV marker mentioned in the workshop.
http://www.bioinnovationllc.com/Page_2.html
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Cell block- microbiopsy-
Role in cytopathologic evaluation/patient management
Critical issues to be considered
Different methods of cell block preparation
Aligning the cells along the cutting surface
Depth of section cutting
Immunophenotyping and cell blocks-
Immunoreactivity interference
Marker for SCIP approach
A few study cases
Q/A session
Outline
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Cell block of cytopathology specimen is
Equivalent to microbiopsy evaluation
the interface between cytology and histopathology
(bridge to histopathology/surgical pathology)
Routine example is Endocervical curretage (ECC),
But without cytology preparation
Cell block with cytology preparation has
Added benefit of excellent cytomorphologic details
in concert with architectural insight
Cyto-histo-pathology
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Role of cell blocks in cytopathologic evaluation/patient management
1. Immunophenotyping
2. Special stains- Mucicarmine, Congo red, organism stains
3. Architectural evaluation-
Trabecular-sinusoidal pattern in HCC,
Hollow or solid proliferation spheres without cores
in carcinoma versus mesothelioma in effusions
Evaluate for invasion
Comparative evaluation with surgical pathology material
E.g- Peritoneal/pelvic washing
Quantification of some features such as mitotic figures
4. Enhanced sampling of FNAB rinses
5. Molecular test
e.g. FISH, CISH, In-situ PCR
6. Archival for future studies
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Critical issues to be considered
Depending on the primary indication,
the method of cell block preparation vary
Multiple variants should be considered
for selecting the method and modifying it as needed for
individual specimens
Specimen type- Fresh versus fixed cells
Cellularity of the specimen
Nature of cell distribution-
predominantly solitary cells versus microfragments/aggregates
Ancillary tests anticipated
Available resources/infrastructure in the lab
Institutional and regional biases
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Different methods of cell block preparation
A. Cell block from specimen with clot or significant sediment- FNAB
B. HistoGel method
C. Gelatin embedding
D. Agar embedding method
E. Plasma-thrombin method
F. Collodion (Celloidin) bag method
G. From scraped material from cytology smears
H. From Millipore filters
I. From cells lifted selectively from the cytology preparation (Kaneko C et al. Diagn Cytopathol 2000;22:117–119)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Processing of FNA aspirate to be submitted to laboratory for Cell block
Let the remaining
aspirate clot in the
syringe for 5 to 7
minutes (slightly
longer than the
clotting time).
1
Transfer the aspirated
formalin with
dislodged cot in to the
specimen container
with 10% formalin
fixative
4
Gently and firmly
remove the plunger of
the syringe .
3
Aspirate 10% formalin
from the container in
which the specimen is
to be submitted for
cell block processing.
This dislodges the clot
from syringe wall.
2
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Protocol for plasma-thrombin method for cell block preparation
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Protocol for cell block preparation with collodion bag
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Protocol for preparing cell blocks with HistoGel
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Aligning the cells along the cutting surface
Depth of section cutting
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Varsegi GM, Shidham V (2009)
Cell Block Preparation from Cytology Specimen with Predominance of Individually
Scattered Cells.
Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Video of JoVE article (8 minutes 15 sec)
At this stage,
please note the URL
and observe the full FREE video in the new internet window
Video of JoVE article (8 minutes 15 sec)
Video article FREE on web as open access at-
http://www.jove.com/index/Details.stp?ID=1316
Or at
http://alturl.com/zkns
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
From:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
From:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Modified from:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
From:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
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) approachShidham & Atkinson
Ch 5. Immunocytochemistry of effusion fl uids: introduction to SCIP approach.
‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
2
3
1
65
4
8
7
Meso
thelial &
infl
am
mato
ry c
ells
2
3
1
65
4
8
7
2
3
1
65
4
8
7
2
3
1
65
4
8
7
2
3
1
65
4
8
7
XM
eta
sta
sis
(no
n-c
arc
ino
ma)
2
3
1
6
54
7
2
3
1
6
54
7
2
3
1
6
54
7
3
1
6
54
7
2
3
1
6
54
7
ZM
eta
sta
sis
(carc
ino
ma)
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
vim
en
tin
Pa
n C
K(M
ixtu
re o
f A
E1
/AE
3
& C
AM
5.2
)
Ca
lre
tin
in
WT
-1
LC
A(C
D45)
[or
PG
M1
(CD
68
)
or
mix
ture
of L
CA
& P
GM
1]
A B C D E
SCIP
approach
Immunocytochemistry
of effusion fluids (continued)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
SCIP
approach
F. CDX2Immunoreactive
nuclear
HE
stained
cell block
section
40X
B. Pan-cytokeratinImmunoreactive
C. LCA (CD45)Non-immunoreactive
A. VimentinNon-immunoreactive
D. CalretininNon-immunoreactive
(Inset {2}-
Mesothelial cell
immunoreactive
nuclear-cytoplasmic)
E. WT-1Non-immunoreactive
(Arrow 2 with inset:
Mesothelial cell-
immunoreactive
nuclear-cytoplasmic)
RM
RM
NC
10X
10X
10X
10X
10X
10X
40X
40X
40X
40X
40X
100X40X
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
RM
RM
NC
Metastatic colonic
adenocarcinoma,
(peritoneal fluid).
35
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
A. Pap smear dx – LSIL,
B. H&E cell block sections,
C. p16 stained cell block sections,
D. biopsy showing CIN II-III
A DCB
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
A. Pap smear dx – HSIL ,
B. H&E cell block section containing “microbiopsies”,
C. p16 stained cell block section showing true nuclear positivity,
D. biopsy showing invasive squamous cell carcinoma .
A DCB
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
12
a b CalretininCalretinin
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.
14
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
a b
c dCongo red stained 10 micron thick sections: Orange yellow birefringence under polarized light.
The color changes to apple green when the axis of polarizer (blue arrows) is changed by 90 degree
Cell block- Fat
pad aspiration
Positive control
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
C. CalretininNon-immunoreactive
(Rare mesothelial
cell [blue arrow] is
immunoreactive
nuclear-cytoplasmic)
D. BerEP4Immunoreactive
E. Estrogen
receptorsImmunoreactive
B. CD68 (PGM1)
Non-immunoreactive
(inflammatory cells are
immunoreactive)
A. Vimentin
Non-immunoreactive
(Mesothelial &
inflammatory cells are
immunoreactive)
‘Subtractive coordinate immunoreactivity pattern’
(SCIP) in cell block sections
20X
20X
20X
20X
20X
40X
40X
40X
40X
40X
NC
RM
NC
NC
Metastatic
mammary adenocarcinoma,
(pleural effusion).
SCIP
approach(continued)
38
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
H&E CD177 vimentin
SMAWS cytokeratin S-100 protein
GIST
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
From: Çoban S et al. BMC Cancer 2004, 4:89
Open access article from:
http://www.biomedcentral.com/1471-2407/4/89
Cell block section of FNA of Hepatocellular Carcinoma
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Study cases- Blocks & slidesBlocks 1-4
1. Peritoneal fluid –solitary tumor cells- with & without AV marker.2. Peritoneal fluid – Metastatic pancreatic adenoca.3. Liquid based cervical cytology specimen- Cell blocks A & B not cut yet.4. Metastatic adenoca with solitary cells – cell block from scant clot
Slides (I - VI)I. Lymphoma (serous cavity effusion)-II. Peritoneal fluid (metastatic pancreatic carcinoma) (See with cell blocks – set #1)
III. p16 immunostaining of cervical cytology LBC specimenIV. Metastatic non-small cell carcinoma (Compare cellularity)V. Metastatic non-small cell carcinoma (pleural fluid) (SCIP approach) VI. Compare H&E stained sections of cell blocks.
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Peer-reviewed,
open access,
teaching material with many pictures.
Hard copy and online availability.
Opportunity for frequent updates
Vinod B. Shidham,
MD, FRCPath, FIAC
Cell BlocksIn
Cytopathology
Milwaukee Art Museum
Detroit
After June 1, 2010