This document outlines Vinod Shidham's presentation on cell blocks. It begins with an introduction to cell blocks and their role in cytopathological evaluation and patient management. It describes various methods for preparing cell blocks from different specimen types and highlights critical issues to consider, such as specimen cellularity and intended ancillary tests. The document discusses aligning cells along the cutting surface and depth of section cutting to improve immunophenotyping on cell blocks. It presents the subtractive coordinate immunoreactivity pattern approach for effusion immunocytochemistry and concludes with several case studies.

1. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Vinod B. Shidham, MD, FRCPath, FIAC
Vice-chair- AP, Professor,
Director of Cytopathology, Cytotechnology School,
Cytopathology fellowship, and
GI Pathology
Executive editor & co-editor-in-chief,
CytoJournal (www.cytojournal.com)
Dept of Pathology, Wayne State University Medical School
Karmanos Cancer Institute & Detroit Medical Center
Detroit, MI 48201, USA
Cell Blocks
for Molecular Tests & Immunocytochemistry ApplicationsFrustrations & Triumphs!
November 9, 2013
(ASC Round Table #11, 12:00 – 1:45 PM)
This presentation is also available at:
http://alturl.com/245zh
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
Disclaimer
VS has indirect financial interest (through spouse) in AV marker referenced.
http://www.bioinnovationllc.com/Home_Page.html
1

2. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Outline
Cell block- microbiopsyRole 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 blocksImmunoreactivity interference
Marker for SCIP approach
A few study cases
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
Cell blocks
Conglomeration of
minute tissue fragments and
isolated cells / small cell groups
in specimens collected or submitted
as suspensions
To processed as
Paraffin embedded tissue sections and
long term archival for future elective evaluations
2

3. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Samples that may be ‘Cell blocked’
Clinical samples
Fluids
Effusion fluids (ascitic, pleural, pericardial, etc)
Other fluids (drainage, cysts, cavities, etc)
Washings (peritoneal, pelvic, bronchial washing)
Exfoliated cells- Cervical cytology specimens
Brushings- Endocervical, bronchial, bile duct etc
Curretages- Endocervical
FNA passes-rinses
Scrapings of cytology smears (stained or unstained)
Any cytology specimen with micro-fragments
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Samples that may be ‘Cell blocked’
(contd)
Research samples
Cell cultures- scrapings of surface grown and suspensions
Other animal experiments related (comparable to clinical samples)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Role of cell blocks in cytopathologic evaluation/patient management
1. Immunophenotyping
2. Special stains- Mucicarmine, Congo red, organism stains
3. Architectural evaluationTrabecular-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
3

4. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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 distributionpredominantly solitary cells versus microfragments/aggregates
Ancillary tests anticipated
Available resources/infrastructure in the lab
Institutional and regional biases
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
J Clin Pachol 1997;50:985-990
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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)
4

5. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Let
the
remaining
aspirate clot in the
syringe for
5 to 7
minutes
(slightly
longer
than
the
clotting time).
1
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
Gently
and
firmly
remove the plunger of
the syringe .
3
Transfer the aspirated
formalin
with
dislodged cot in to the
specimen
container
with 10% formalin
fixative
4
Processing of FNA aspirate to be submitted to laboratory for Cell block
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Protocol for plasma-thrombin method for cell block preparation
Shidham & Epple
Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)
5

6. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
Automated cell block system
http://www.cellientsystem.com/
6

7. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Wagner DG et.
Diagn Cytopathol 2011 Oct;39(10):730-6
Vinod B. Shidham, MD, FRCPath, FIAC
Immunocytochemistry & Vice chair- AP
Professor of imprint smears
(continued)
vshidham@med.wayne.edu
Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21.
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
However, all these methods face
the critical challenge due
unpredictable nature of the section cutting
of paraffin blocks with scant scattered cells
7

8. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Microfragments
& cells in
specimen
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Aligning the cells along the cutting surface
Depth of section cutting
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
8

9. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Video article is available FREE on web in
open access athttp://www.jove.com/index/Details.stp?ID=1316
Video of JoVE article (8 minutes 15 sec)
Video of JoVE article (8 minutes 15 sec)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
From:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
9

10. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
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
Professor & Vice chair- AP
vshidham@med.wayne.edu
From:
Varsegi GM, Shidham V (2009)
Journal of Visualized Experiments
(JoVE) 2009 Jul 21;(29). pii: 1316.
doi: 10.3791/1316. PMID: 19623160
10

11. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Immunophenotyping and cell blocksFactors affecting immunoreactivityLoss, reduction, or
enhancement of antigen immunoreactivity
Exposure to different reagents and fixative(s)
Temperature
Storage of specimen with or without fixative
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Subtractive Coordinate Immunoreactivity Pattern (SCIP) approach
Shidham & 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
Professor & Vice chair- AP
7
6
7
6
2
5
3
7
4
5
1
4
5
5
7
1
4
5
3
3
2
1
5
7
3
4
5
1
5
7
6
7
6
7
2
6
2
3
4
1
Immunocytochemistry
of effusion fluids
(continued)
7
5
2
3
6
4
SCIP
approach
8
2
8
6
6
3
6
8
3
2
7
5
4
E
WT-1
1
5
7
4
1
D Calretinin
4
[or PGM1(CD68)
or mixture of LCA
& PGM1]
7
5
3
3
C LCA(CD45)
1
5
7
4
1
4
2
1
6
3
6
2
8
2
3
1
B Pan CK
(Mixture of AE1/AE3
& CAM5.2)
7
4
5
3
1
6
6
3
1
4
4
2
2
4
6
8
6
3
1
2
1
A vimentin
Mesothelial & X
inflammatory cells
7
5
Metastasis
(carcinoma)
2
Y
Z
Metastasis
(non-carcinoma)
vshidham@med.wayne.edu
11

12. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Study cases
12

13. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
CD10
pCEA
(+control)
pCEA
vshidham@med.wayne.edu
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
H&E
CD177
vimentin
GIST
WS cytokeratin
S-100 protein
SMA
13

14. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Shidham VB et al. J Vis Exp. (2010) 44.
http://www.jove.com/index/Details.stp?ID=1747
Cell block- Fat
pad aspiration
a
b
c
d
Positive control
Congo 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
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
35
vshidham@med.wayne.edu
A. Vimentin
Non-immunoreactive
NC
NC
10X
40X
B. Pan-cytokeratin
Immunoreactive
NC
NC
10X
40X
C. LCA (CD45)
Non-immunoreactive
SCIP
approach
NC
NC
10X
40X
D. Calretinin
Non-immunoreactive
(Inset {2}RM
Mesothelial cell
immunoreactive
nuclear-cytoplasmic) RM
NC
10X
NC
40X
E. WT-1
Non-immunoreactive
(Arrow 2 with inset:
Mesothelial cellimmunoreactive
RM
nuclear-cytoplasmic)
HE
stained
cell block
section
RM
NC
NC
10X
40X
Shidham & Atkinson.
Cytopathologic Diagnosis of
Serous Fluids
NC
F. CDX2
Immunoreactive
nuclear
NC
NC
40X
Metastatic colonic
adenocarcinoma,
(peritoneal fluid).
10X
40X
Elsevier.
100X
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
SCIP approach
HE
stained
cell block
section
Metastatic colonic
adenocarcinoma,
(peritoneal fluid).
Shidham & Atkinson.
Cytopathologic Diagnosis of
Serous Fluids
Elsevier.
14

15. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
2
a
Calretinin
b
1
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.
‘Subtractive coordinate immunoreactivity pattern’
(SCIP) in cell block sections
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
A. Vimentin
Non-immunoreactive
(Mesothelial &
inflammatory cells are
immunoreactive)
vshidham@med.wayne.edu
20X
40X
Metastatic
mammary adenocarcinoma,
(pleural effusion).
B. CD68 (PGM1)
Non-immunoreactive
(inflammatory cells are
immunoreactive)
20X
40X
RM
C. Calretinin
Non-immunoreactive
(Rare mesothelial
cell [blue arrow] is
immunoreactive
nuclear-cytoplasmic)
20X
40X
20X
40X
D. BerEP4
Immunoreactive
SCIP
approach
NC
(continued)
NC
E. Estrogen
receptors
NC
Immunoreactive
40X
20X
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
A
B
C
D
A. Pap smear dx – LSIL,
B. H&E cell block sections,
C. p16 stained cell block sections,
D. biopsy showing CIN II-III
15

16. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
A
B
C
D
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 .
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Am J Clin Pathol 2011;136:564-571
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
16

17. Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
open access,
teaching material with many pictures.
Hard copy and online availability.
Opportunity for frequent updates
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
vshidham@med.wayne.edu
17