using cytology specimens for immunohistochemistry
TRANSCRIPT
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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
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Disclosures
! Don’t have any.
! I’m just a glass pusher – what do you expect.
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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
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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
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IHC Surgical Pathology vs. Cytology ! IHC Cytology
! Marked variaOons ! FixaOon ! Slide preparaOons ! Pre-‐IHC manipulaOon
! Prior staining ! Tissue fragment recovery from slide
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“Substrates” for IHC in Cytology ! Cytology slides
! Direct smears ! Cytocentrifuge slides ! ThinPrep slides ! SurePath slides ! Filter preparaOon slides
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“Substrates” for IHC in Cytology ! Cell block preparaOons
! Fresh sample ! Pre-‐fixed sample ! Recovered cyto-‐prep samples
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“Substrates” for IHC -‐ Cytology slides
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“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
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“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
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“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
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“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
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“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
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“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
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“Substrates” for IHC -‐ Cytology slides ! AlternaOve to staining cytology slide
! Cell/Ossue recovery and cell block producOon
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“Substrates” for IHC – Cell Blocks
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“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
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“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
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“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
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PreparaOon of Cell Blocks
0
1
2
3
4
5
6
7
8
FNA Needle Rinse Fluid
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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
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Does Pre-‐fixaOon Ma6er?
Cancer Cytopathol. 2014 Feb;122(2):145-‐52
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PreparaOon of Cell Blocks
0
2
4
6
8
10
12
Tissue Aggregation Medium
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PreparaOon of Cell Blocks
0
5
10
15
20
25
30
10% Formalin B-Plus fixative
Cell Block Fixative
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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
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PreparaOon of Cell Blocks
0
5
10
15
20
25
<72 hours >72 hours
Maximum Duration of Fixation
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European FederaOon of Cytology SocieOes (EFCS) Inquiry (Cytopathol 2011;22:238–242) ! On-‐line survey, 2008
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College of American Pathologists (Arch Pathol Lab Med. doi: 10.5858/arpa.2013-‐0259-‐CP)
! Voluntary survey, fall 2009 – 818 responses
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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
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UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
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UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
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UK NEQAS ICC -‐ Cytology Module (Cytopathol 2011;22:230-‐237)
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Where do we go from here?
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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
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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
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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
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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
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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
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More Challenging Issues ! Cytology slides as substrates
! How is IHC being used? ! Substrate specific controls? ! Historic validaOon?
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Thank you
UNIVERSITY of TORONTO
Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto [email protected]
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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
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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