Vinod B. Shidham is the executive editor and co-editor-in-chief of CytoJournal. He is a professor and director of cytopathology fellowship training at Wayne State University School of Medicine, Karmanos Cancer Center, and Detroit Medical Center. The document appears to be from a CME on cell block making that discusses techniques for concentrating specimens, embedding cell blocks, immunophenotyping from cell blocks, and case studies.

1. Vinod B. Shidham, MD, FRCPath, FIAC
Executive editor & co-editor-in-chief, CytoJournal (www.cytojournal.com)
Professor, Vice-chair- AP, Director of GI pathology & Cytopathology
Director of Cytopathology Fellowship Training Program
Department of Pathology
Wayne State University School of Medicine, Karmanos Cancer Center, &
Detroit Medical Center
Kressege Campus- Old Hutzel Hospital (Dept of Cytology- Ground Floor),
4707 St. Antoine Blvd
Detroit, MI 48201
vshidham@med.wayne.edu
VAPM- Nagpur, India
4th Smt. Sunanda B Shidham memorial CME
CellBlockistry:
Chemistry & Art of Cell Block Making

6. Nano
Need Centrifuges with free swinging rotors

7. AV marker
Bottom view
Concentrated specimen sediments
in the wells
Bottom view
Nano- NextGen CelBloking Gel disc

8. Nano- NextGen CelBloking Gel disc with wells
AV marker Concentrated specimen sediments
in the wells

10. From Pack #1
From Pack #2

15. Embedding instructions

16. Cutting the paraffin embedded cell block

18. AV marker
Courtesy: AV BioInnovation

19. AV marker
Courtesy: AV BioInnovation

20. AV marker AV marker
Courtesy: AV BioInnovation

21. Cellular specimen NextGen CellBlocking- Micro
Cellular specimen
2X
20X
Conventional Method
NextGen CellBlockingDevice
(Based on Shidham principle) AV BioInnovation
http://www.jove.com/index/Details.stp?ID=1316
NextGen CellBlocking- Nano
Diluted cellular specimen
Courtesy: AV BioInnovation

22. Nano Cell Block section

23. Micro Cell Block section

24. For specimen with predominance of blood
Specimens with predominance of blood may not
generate good cell block sections with diagnostic cells.
This is especially true for many FNA needle rinses and
Hemorrhagic body fluids.
Although the specimen may have diagnostic cells, they
may not align along the cutting surface even with
Nano NextGen CelBloking units.

25. Specimen with predominance of blood
Cutting surface
Red blood cells
settling down
along the
bottoms
Diagnostic cells
settling above
the red blood
cells like
buffy coat
Blood rich
specimen
Blood rich
specimen
Centrifuge
3 mnts

26. For specimen with predominance of blood
For better results, the red blood cells should be lysed by
the method which will not affect the immunoprofile of the
diagnostic cells / tissue.
Thus using the reagent for lysing the red blood cells
during flow cytometry may be used.
Avoid using reagents with acidic lysing agents such as
with acetic acid.
BloodLyzTM is suitable to lyse the contaminant red blood
cells without compromising the immunoprofile.

27. Lysing the contaminant blood
BloodLyzTM
Add blood rich
specimen
Red blood cells
in the specimen
are lysed without
affecting the
diagnostic cells
10
mnts
Centrifuge
3 mnts
Sediments with diagnostic cells
concentrated along the bottom.
Use this for making cell block
after discarding the supernatant.
Supernatant with
hemolysed red blood cells.
*
*The red blood cells can only be lysed if the specimen is fresh
or collected in isotonic medium without any fixative.
The lysis may not work effectively with specimens collected in
fixatives including CytoLyte and Sacamano fluid etc.

28. Immunophenotyping and cell blocks

29. 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

30. Subtractive Coordinate Immunoreactivity Pattern (SCIP) approach
Shidham & Atkinson
Ch 5. Immunocytochemistry of effusion fluids:
introduction to SCIP approach.
‘Cytopathologic Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders Company)

31. 2
3
1
6
5
4
8
7
Mesothelial&
inflammatorycells
2
3
1
6
5
4
8
7
2
3
1
6
5
4
8
7
2
3
1
6
5
4
8
7
2
3
1
6
5
4
8
7
XMetastasis
(non-carcinoma)
2
3
1
6
5
4
7
2
3
1
6
5
4
7
2
3
1
6
5
4
7
3
1
6
5
4
7
2
3
1
6
5
4
7
ZMetastasis
(carcinoma)
21
5
4
3
7
6
21
5
4
3
7
6
21
5
4
3
7
6
21
5
4
3
7
6
21
5
4
3
7
6
Y
vimentin
PanCK
(MixtureofAE1/AE3
&CAM5.2)
Calretinin
WT-1
LCA(CD45)
[orPGM1(CD68)
ormixtureofLCA
&PGM1]
A
B
C
D
E
SCIP
approach
Immunocytochemistry
of effusion fluids
(continued)

32. Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)

33. Courtesy: AV BioInnovation
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’ Elsevier (W. B. Saunders Company); 2007. Ch 5.

34. Courtesy: AV BioInnovation
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’ Elsevier (W. B. Saunders Company); 2007. Ch 5.

35. Shidham & Atkinson
Ch 5. ‘Cytopathologic
Diagnosis of Serous Fluids’
Elsevier (W. B. Saunders
Company)

36. Study cases

38. pCEA
pCEA
(+control)
CD10

39. H&E CD177 vimentin
SMAWS cytokeratin S-100 protein
GIST

40. a b
c d
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
Cell block- Fat
pad aspiration
Positive control
Shidham VB et al. J Vis Exp. (2010) 44.
http://www.jove.com/index/Details.stp?ID=1747

41. SCIP
approach
F. CDX2
Immunoreactive
nuclear
HE
stained
cell block
section
40X
B. Pan-cytokeratin
Immunoreactive
C. LCA (CD45)
Non-immunoreactive
A. Vimentin
Non-immunoreactive
D. Calretinin
Non-immunoreactive
(Inset {2}-
Mesothelial cell
immunoreactive
nuclear-cytoplasmic)
E. WT-1
Non-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
Shidham & Atkinson.
Cytopathologic Diagnosis of
Serous Fluids
Elsevier.

42. SCIP approachHE stained
cell block section
Metastatic colonic
adenocarcinoma,
(peritoneal fluid).
Shidham & Atkinson.
Cytopathologic Diagnosis of
Serous Fluids
Elsevier.

43. 1
2
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.

44. C. Calretinin
Non-immunoreactive
(Rare mesothelial
cell [blue arrow] is
immunoreactive
nuclear-cytoplasmic)
D. BerEP4
Immunoreactive
E. Estrogen
receptors
Immunoreactive
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)

45. 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

46. 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

47. Am J Clin Pathol 2011;136:564-571