Using Cytology Specimens for Immunohistochemistry
Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto [email protected]
UNIVERSITY of TORONTO
Disclosures
! Don’t have any.
! I’m just a glass pusher – what do you expect.
Learning Objec-ves At the compleOon of this session the parOcipants will have acquired an understanding of: ! Suitability of cytologic samples for molecular tesOng ! The variety of cytologic substrates for molecular tesOng ! How different cytologic sampling techniques differ in
their potenOal as substrates for molecular tesOng ! The impact of pre-‐analyOcal processing on suitability for
molecular tesOng ! LimitaOons of cell block producOon
IHC Surgical Pathology vs. Cytology ! IHC in Surgical Pathology
! Standardized ! 10% Neutral Buffered Formalin (NBF) fixaOon ! Paraffin embedded ! Tissue secOons
! VariaOons in ! DuraOon of ischemia prior to fixaOon ! DuraOon of fixaOon ! IHC techniques
IHC Surgical Pathology vs. Cytology ! IHC Cytology
! Marked variaOons ! FixaOon ! Slide preparaOons ! Pre-‐IHC manipulaOon
! Prior staining ! Tissue fragment recovery from slide
“Substrates” for IHC in Cytology ! Cytology slides
! Direct smears ! Cytocentrifuge slides ! ThinPrep slides ! SurePath slides ! Filter preparaOon slides
“Substrates” for IHC in Cytology ! Cell block preparaOons
! Fresh sample ! Pre-‐fixed sample ! Recovered cyto-‐prep samples
“Substrates” for IHC -‐ Cytology slides
“Substrates” for IHC -‐ Cytology slides ! Direct smears / Fresh Cytocentrifuge Preps
Air-‐dried/Methanol Fixed
Unstained Romanowsky stained
IHC De-‐stain
Wet/Ethanol Fixed/Spray Fix
Unstained
Papanicolaou stained
IHC De-‐stain
“Substrates” for IHC -‐ Cytology slides ! PreservaOves commonly used:
CytoRich Red CytoRich Blue Cytospin Collec-on Fluid
Saccomanno Fixa-ve
Ethanol -‐ 44% 40% 40%
Methanol 7-‐13% 5% 2% 2%
Isopropanol 10-‐30% 2% +/-‐ 2%
Ethylene Glycol 5-‐10% -‐ -‐ -‐
Polyethylene Glycol -‐ 1% 2% 2%
Formaldehyde <1% -‐ -‐ -‐
Others FD&C red No. 40 ? Methylene blue Methylene blue + FD&C yellow No. 5 Proprietary PreservaOve
“Substrates” for IHC -‐ Cytology slides ! PreservaOves commonly used:
25% Ethanol (“add” equal volume
of 50% ETOH) CytoLyt PreservCyt SurePath
Fixa-ve
Ethanol 25% -‐ -‐ 20%
Methanol -‐ 20-‐50% 30-‐60% 1%
Isopropanol -‐ -‐ -‐ 1%
Ethylene Glycol -‐ -‐ -‐ -‐
Polyethylene Glycol -‐ -‐ -‐
Formaldehyde -‐ -‐ -‐ -‐
Others Denaturing agent Proprietary agents Proprietary agents Proprietary agents
“Substrates” for IHC -‐ Cytology slides ! PreservaOves commonly used:
Cyto-‐”Sprays” Modified Carnoy’s 95% Ethanol
Ethanol 40-‐50% 60-‐86% 95%
Methanol <5% -‐ -‐
Isopropanol 1-‐6% -‐ -‐
Ethylene Glycol 1-‐10% -‐ -‐
Polyethylene Glycol -‐ -‐ -‐
Formaldehyde -‐ -‐ -‐
Others Propellent (Isobutane) 10% AceOc Acid Denaturing agent
“Substrates” for IHC -‐ Cytology slides ! Does it work? – Yes, qualitaOvely and
quanOtaOvely, but; ! Studies occasionally contradict one another ! Marked variaOons in IHC protocols
! Air-‐dried ! RehydraOon / no rehydraOon
! Air-‐dried and alcohol fixed ! Post-‐fixaOon – none / formalin / formalin + ETOH / acetone + methanol / etc.
! Epitope retrieval – yes / no
“Substrates” for IHC -‐ Cytology slides ! Does it work? – Yes, qualitaOvely and
quanOtaOvely, but; ! Lack of opOmizaOon of IHC protocols for
cytology slides ! Lack of substrate specific controls
“Substrates” for IHC -‐ Cytology slides ! AlternaOve to staining cytology slide
! Cell/Ossue recovery and cell block producOon
“Substrates” for IHC – Cell Blocks
“Substrates” for IHC – Cell Blocks ! StarOng condiOons of the sample
! Fresh vs. preserved / fixed ! Age of fresh samples
! FixaOves used
! ManipulaOons of the sample prior to formalin fixaOon ! Rinsing of the sample (i.e. effusions) ! Recovery of sample from a cytology slide
“Substrates” for IHC – Cell Blocks Fresh Sample
Rinsing of Sample
Binding Agent
Fixation of Sample
Histologic Processing of Block
Fixation of Sample
Binding Agent
Fixation of Sample
Fresh Sample in Saline
Binding Agent
“Substrates” for IHC – Cell Blocks ! An informal SurveyMonkey survey conducted of
Cytology labs Nov-‐Dec. 2012 ! Distributed via the Canadian Society of
Cytopathology email distribuOon list ! 27 respondents
! IdenOfy pracOces in preparaOon of cell blocks ! Fine needle aspiraOons ! Other non-‐gynecologic samples
PreparaOon of Cell Blocks
0
1
2
3
4
5
6
7
8
FNA Needle Rinse Fluid
PreparaOon of Cell Blocks
0
2
4
6
8
10
12
14
None CytoLyt PreservCyt CytospinCollection
Fluid
SaccomannoFluid
PlasmaLyte +CytoLyt
50% alcohol
Non-Gyne Preservative
Does Pre-‐fixaOon Ma6er?
Cancer Cytopathol. 2014 Feb;122(2):145-‐52
PreparaOon of Cell Blocks
0
2
4
6
8
10
12
Tissue Aggregation Medium
PreparaOon of Cell Blocks
0
5
10
15
20
25
30
10% Formalin B-Plus fixative
Cell Block Fixative
PreparaOon of Cell Blocks
0
1
2
3
4
5
6
7
8
9
<4 hours 4 - 6 hours >6 hours >8 hours 24 hours Uncertain
Minimum Duration of Fixation
PreparaOon of Cell Blocks
0
5
10
15
20
25
<72 hours >72 hours
Maximum Duration of Fixation
European FederaOon of Cytology SocieOes (EFCS) Inquiry (Cytopathol 2011;22:238–242) ! On-‐line survey, 2008
College of American Pathologists (Arch Pathol Lab Med. doi: 10.5858/arpa.2013-‐0259-‐CP)
! Voluntary survey, fall 2009 – 818 responses
UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
! UK external quality assessment of immunocytochemistry on cytology samples ! 140 labs in UK (75), Switzerland (8), Portugal (8),
Norway (5), Canada (3), Australia (3), US (2), etc.
! Panel of 4 experts “grade” cytospins sent for tesOng as well as in-‐house control slide
! Cytospin Methanol fixed (12-‐16 hr) 3%PEG
UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
Where do we go from here?
What Can We Do – First Steps ! Include a gross descripOon on all cytology
reports: ! Detailing handling of the sample
! CondiOon on receipt (fresh/preserved/fixed) ! ManipulaOons of sample (washes with what?)
! FixaOon (with what) ! ? Binding agent for block producOon ! Stains used
What Can We Do – First Steps ! Why a gross descripOon?
! In-‐house tesOng ! Protocol changes over Ome
! Out-‐of-‐house tesOng ! ConsultaOons ! AddiOonal invesOgaOons upon referral ! Reference lab tesOng
What Can We Do – First Steps ! Approach standardizaOon of cell blocks
! Gravitate towards universal NBF fixaOon ! Must accept two streams
! Fresh Formalin ! “Alcohol” pre-‐fixaOon Formalin
! Fresh in cytology and surgical pathology are slightly different: ! Surg Path – significant hypoxic period prior to fixaOon
! Devascularized / penetraOon of formalin ! Cyto – “immediate” fixaOon vs. collecOon in physiologic saline prior to fixaOon
What Can We Do – First Steps ! Approach standardizaOon of cell blocks
! Do not use fixaOves other than alcohol or formalin
! ? Standard NBF fixaOon prior to histologic processing
! Standardize minimum fixaOon duraOon ! ? Fix fresh samples prior Ossue aggregaOon ! ? Standardize Ossue aggregaOon media
What Can We Do – First Steps ! Approach standardizaOon of cell blocks
! PosiOve controls ! Validate equivalence of Cyto cell block & Surg Path handling ! Use of Surg Path controls or ! Use of Cyto specific controls
More Challenging Issues ! Cytology slides as substrates
! How is IHC being used? ! Substrate specific controls? ! Historic validaOon?
Thank you
UNIVERSITY of TORONTO
Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto [email protected]
Which of the following fixaOves appears to have the more deleterious effect on immunohistochemical staining?
1. CytoRich Red 2. CytoLyt 3. Formalin 4. Acetone 5. Ethanol
World wide, what substrates are used of immunohistochemical staining for cytology samples?
1. Air-‐dried direct smears 2. Formalin-‐fixed cell blocks 3. ThinPrep prepared slides 4. Wet-‐fixed cytocentrifuge slides 5. All of the above
