cell block on

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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, USAvshidham@med.wayne.edu

Cell Blocks

In Cytopathology(Workshop# 5 & 43)

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Shidham & Epple

Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’

Elsevier (W. B. Saunders Company)

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Aligning the cells along the cutting surface

Depth of section cutting

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Shidham & Atkinson

Ch 5. ‘Cytopathologic

Diagnosis of Serous Fluids’

Elsevier (W. B. Saunders

Company)

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Shidham & Atkinson

Ch 5. ‘Cytopathologic

Diagnosis of Serous Fluids’

Elsevier (W. B. Saunders

Company)

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Study cases

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

H&E CD177 vimentin

SMAWS cytokeratin S-100 protein

GIST

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Q/A

Vinod B. Shidham,

MD, FRCPath, FIAC

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Cell BlocksIn

Cytopathology

Milwaukee Art Museum

vshidham@mcw.edu

Detroit

After June 1, 2010

vshidham@med.wayne.edu