Using cytology specimens for immunohistochemistry

transcript

Using Cytology Specimens for Immunohistochemistry

Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto sco6.boerner@uhn.ca

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

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

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

FNA Needle Rinse Fluid

None CytoLyt PreservCyt CytospinCollection

SaccomannoFluid

PlasmaLyte +CytoLyt

50% alcohol

Non-Gyne Preservative

Does Pre-‐fixaOon Ma6er?

Cancer Cytopathol. 2014 Feb;122(2):145-‐52

Tissue Aggregation Medium

10% Formalin B-Plus fixative

Cell Block Fixative

<4 hours 4 - 6 hours >6 hours >8 hours 24 hours Uncertain

Minimum Duration of Fixation

<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)

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

!  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

!  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 sco6.boerner@uhn.ca

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

Using cytology specimens for immunohistochemistry

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