Immunohistochemistry on Cell Block specimens Effects of fixation and time
Tom Thomson MD, FRCPC
Laboratory staff
BC Cancer Agency Clinical
Laboratory
Vancouver, British
Columbia
CIQc/ACDIHC Symposium June 20-21, 2014 St. John’s, Newfoundland
Faculty Disclosures
• Dr Thomas Thomson has no relevant financial
relationships with commercial interests to disclose.
• Funding for the reported study entirely from the
BC Cancer Agency, Vancouver Cancer Center
laboratory as a Quality Assurance project
• Approved by the UBC ethics review board
• At the end of this presentation, participants will increase awareness of the importance of pre-analytic variables such as cell preservation and time on immunohistochemistry on cell block preparations.
• This session will present unpublished information on an ongoing study of immunostains on cell blocks of body cavity fluids and current observations.
Learning Objectives
Outline
• Overview of our fluid control project
• Summary of findings to date
• Impact on our practice
Preamble
Prior to 2013 the Vancouver Cancer Center laboratory recommendation was that body cavity fluids be preserved in equal volumes of 50% ethanol
This is the standard method of preservation of fluids from texts of cytology (Koss, Keebler)
A false negative stain for Estrogen receptor on pleural fluid lead to this study and change in our policy
Cell blocks are increasingly important
Diagnosis: confirmatory special stains
- Results may be essential for staging
Ancillary studies: IHC and Molecular
May be the only or best material available from a patient with advanced disease.
Despite numerous potential variables in cell block preparations, there is remarkably little literature on the effects of pre analytic variables on immunohistochemical stains.
The Study - a work in progress
• Examine fixation variables, type and time
• Create a bank of cell blocks for controls
• Establish a continuous assessment process
and renewal of cell block controls
Source materials
• Body cavity fluids from VCC clinic patients
– Partner with clinicians and nursing staff
– Ethics approval
– Protocol and Consent process organized
Variables
Aggregation Medium: Albumin vs. Histogel®
Preservatives: Ethanol, CytoLyt®, Formalin
Fixatives: all blocks were formalin fixed
Time interval from preservation to block:
Same day – Immediate
Same day – delayed 4-6 hours
Overnight
Method
Using the same protocols as IHC on tissue, compare
stain results on cell blocks prepared with variable
time exposure to different cell preservatives using
several antibodies.
Note:
- does not eliminate stain variation in tumors
- does not eliminate false positives and negatives
Media Reagents Equipment Supplies
N/
A
Diff Quik
Stain
PAP stain
and required
solutions
Methanol
95% Ethanol
Formalin
CytoLyt,
PreservCyt
Albumin
Biological Safety Cabinet
(BSC)
Centrifuge
Cytocentrifuge
Tissue Tek Drs Stainer
Surgipath Automated
Coverslipper
Timer
Fluid control spreadsheet:
H:\Lab_Medicine\Diagnos
tic_Cytology\QA and
OUTCOME\FLUID
CONTROLS
Patient consent form
Personal protective
apparel. Refer to:
CASM_001_4002 Use of
Personal Protective
Apparel
10x 50mL centrifuge tube
3x 15mL centrifuge tube
Solvent resistant pen
Glass slides
Liquid waste disposal
container
Staining basket
Disposable plastic pipette
Coverglass #1 – 24x50
Preparing Immunohistochemistry Controls for Cytology Cell Block Specimens
•Purpose: This procedure provides instructions for the preparation of immunohistochemistry controls on cytology cell block material.
•Samples: Body cavity fluids: 500 – 1000 ml required
•Materials:
Standard Operating Procedure created
Protocol in brief
• Daily check of patient list by cytology clerk • Pathologist identifies suitable specimens • Fluid collected and processed same day • Diff Quik stain to assess suitability • Cell blocks prepared – anonymous label
• Cell blocks reviewed for quality
• Histology cuts sections and performs selected stains
• Stains interpreted – intensity and percent
Specimens (to date)
• Collected: 47 (Peritoneal 41, Pleural 6)
• IHC stained: 32
– 15 insufficient cells or no tumor
(11 cases of HCC and 3 NET - no tumor)
• Approved but cancelled: 100
– no consent , procedure cancelled, no tumor,
Tumors Blocks IHC Stained
Serous carcinoma 16 14
Lung adenocarcinoma 4 4
Renal cell carcinoma 4 4
Breast 3 2
Carcinoid 3 0
Colorectal 3 2
Gastric 2 2
Clear Cell Ovary 1 1
Melanoma 1 1
Immunostains
• Epithelial: MOC31, BerEp4
• Mesothelial: Calretinin, CK5/6, D2-40 (podoplanin)
• Differential:
– Nuclear: ER, WT1, PAX8, CDX2, TTF1, S-100
– Cytoplasmic: Keratin 7, 20, EMA, CD10, CAIX, Mammoglobin, HMB-45(melanosome), Melan-A
Staining Platform
• Benchmark XT (Ventana)
• Detection system:
• Ventana Ultraview Universal DAB Kit
• Switched Feb 4, 2014 to Optiview DAB IHC Detection Kit (specimens FT43 and later)
• Epithelial Moc31 and BerEp4 are manual
ANTIBODY DIL VENDOR Cat # CLONE
Ber-EP4 1:25 Dako Canada Inc M0804 Ber-EP4
CA IX 1:1000 Novus Biologicals NB100-417SS Polyclonal
Calretinin Pre-diluted Ventana 790-4467 SP65
CD163 1:100 Leica - Novocastra NCL-CD163 10D6
CDX2 DAK 1:20 Dako Canada Inc M3636 DAK-CDX2
CK20** 1:50 Dako Canada Inc M7019 Ks20.8
CK5/6 1:50 Dako Canada Inc M7237 D5/16B4
CK7 1:50 Dako Canada Inc M7018 OV-TL 12/30
D2-40 1:100 Dako Canada Inc M3619 D2-40
EMA 1:25 Dako Canada Inc M0613 E29
ER Pre-diluted Ventana 790-4325 SP1
GCDFP-15 1:50 Dako Canada Inc M3638 23A3
HMB45 1:100 Dako Canada Inc M0634 HMB45
Mammaglobin 1:100 Dako Canada Inc M3625 304-1A5
Melan-A 1:25 Dako Canada Inc M7196 A103
MOC-31 1:50 Dako Canada Inc M3525 MOC-31
Napsin A 1:200 Leica - Novocastra NCL-L-Napsin A IP64
p53 1:100 Dako Canada Inc M7001 DO-7
PAX8 1:250
Cedarlane/ProteinTech
Group Inc 10336-1-AP Polyclonal
S100 1:3000 Dako Canada Inc Z0311 Polyclonal
TTF-1 1:100 Leica - Novocastra NCL-L-TTF-1 SPT24
WT1 1:10 Leica - Novocastra NCL-L-WT1-562 WT49
**CK20_May 29, 2014 Ventana clone SP33 Pre-diluted antibody
Fixed within an hour 95% EtOH Fixative (a) Albumin CB (i)
Processed same day HistoGel CB (ii)
Formalin Fixative (b) Albumin CB (i)
HistoGel CB (ii)
Cytospin (iii)
Cytolyte Fixative (c) Albumin CB (i)
HistoGel CB (ii)
Cytospin (iii)
Fixed within an hour 50/50 EtOH Overnight (d) Albumin CB (i)
Processed Next Day
HistoGel CB (ii)
50/50/ Formalin Overnight (e) Albumin CB (i)
HistoGel CB (ii)
50/50 Cytolyte Overnight (f) Albumin CB (i)
HistoGel CB (ii)
ORIGINAL PROTOCOL
Albumin-Serous Ca FT8– WT1 Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
HistoGel-Serous Ca FT8– WT1 Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
Block Time Prep FIXATION Time
A Same Day Al-CB Ethanol 95% 1 hr
B Same Day Al-CB Formalin 1 hr
C Same Day Al-CB Cytolyt 1 hr --> PreservCyt 10min 1 hr
D Overnight Al-CB Ethanol 50% Overnight
E Overnight Al-CB Formalin Overnight
F Overnight Al-CB Cytolyt --> PreservCyt 10min Overnight
G Same Day-
Timed Al-CB Cytolyt 4-6 hrs --> PreservCyt 4 hr
H Same Day Cytospin Formalin --> Formalin Cytospins 1 hr
I Same Day Cytospin Cytolyt --> PreservCyt 10min cytospins 1 hr
J Overnight Cytospin Cytolyt --> PreservCyt 10min cytospins Overnight
REVISED CURRENT PROTOCOL
Morphology Lung Adenocarcinoma FT36
FT03 FT04 FT42 FT43 FT44 FT45 FT48 FT04 FT 40 FT 41 FT42 FT43 FT44 FT45 FT48 FT49
TIME_FIX
PREP
BerEp4 BerEp4 BerEp4 BerEp4 BerEp4 BerEp4 BerEp4 MOC31 MOC31 MOC31 MOC31 MOC31 MOC31 MOC31 MOC31 MOC31
SDy_Eth Al-CB 3/100 3/100 0/0 3/100 3/100 2/100 0/0 3/100
SDy_For Al-CB 3/100 3/100 0/0 3/100 3/100 3/100 0/0 3/100
SDy_Cyt Al-CB 3/100 3/100 0/0 3/100 3/100 3/100 0/0 3/100
ON_Eth Al-CB 3/100 3/100 0/0 3/100 3/100 2/100 0/0 3/100
ON_For Al-CB 3/100 3/100 0/0 3/100 3/100 3/100 2/60 3/100 2/80 2/100 0/0 3/100 3/100 3/100 3/60 3/100
ON_Cyt Al-CB 2/100 3/100 0/0 3/100 3/100 2/90 1/30 3/100 2/80 0/0 3/100 3/100 3/100 2/80 3/100
SDy_Cyt_4-6h Al-CB 0/0 3/100 3/100 0/0 3/100
SDy_For Cspn 3/100
SDy_Cyt Cspn 2/90
ON_Cyt Cspn 2/90
BerEp4 MOC31
EPITHELIAL MARKERS
Epithelial markers; Serous Carcinoma; FT45
Preserved and processed same day:
MOC31 BerEp4
FT04 FT13 FT 40 FT 41 FT42 FT45 FT04 FT13 FT 40 FT45 FT 40 FT42 FT43 FT45
TIME_FIX PREP Calret Calret Calret Calret Calret Calret CK5/6 CK5/6 CK5/6 CK5/6 D2-40 D2-40 D2-40 D2-40
SDy_Eth Al-CB 3/100 3/* FP 3 2<1 3/100 3/100 3 0/0
SDy_For Al-CB 3/100 3/100 3/* FP 3/100 3/* FP 3/100 3 3/* FP 3/100 3/*
SDy_Cyt Al-CB 3/100 0/0* 3 1/10 3/100 3/* FP 3 3/*
ON_Eth Al-CB 3/100 0/0* 0/0* 3/* 1/* 2/100 2/* 2/*; FP 3/* 0/*
ON_For Al-CB 3/100 3/90 3/* FP 3 3/* FP 3/100 3/80 3/* FP 3/80 3 0 3/*
ON_Cyt Al-CB 2/* FP 3 0/0 3/100 1/10; FP 2/* 0/*
SDy_Cyt_4-6h Al-CB 0 <1/* 0/0; FP 2/20; FP 3 3/*
SDy_For Cspn 3/* FP
SDy_Cyt Cspn 0
ON_Cyt Cspn 0
Calretinin CK5/6 D2-40
* = Rare cells
Mesothelial
Mesothelial markers; expected results Formalin overnight Lung Adenocarcinoma; FT38
Mesothelial markers - Pitfalls False positive tumor staining with Calretinin and CK5/6
but not with D2-40
Formalin same day, Serous Carcinoma; FT45
Mesothelial markers – Pitfalls Loss of Calretinin with Cytolyt but retained with D2-40
FT42 History-Colon
Adenocarcinoma
No tumor
Fixative PREP FT08 FT09 FT15 FT16 FT17 FT19 FT20 FT21 FT27 FT34 FT35 FT43 FT45
Ser Ser Ser Br Ser Ser Ser Ser Ser Ser Ser Ser Ser
ER ER ER ER ER ER ER ER ER ER ER ER
SDy_Eth Al-CB 3/90 2-3/50 3, 100 2, 25 2/20
SDy_For Al-CB 3/90 3/80 2/* 3/100 3/100 3/* 3/100 3/90 3, 100 3/100 3/100
SDy_Cyt Al-CB 1-2<50 2/30 2/* 3/* 2/* 0/0 2-3/100 2/70 1-2<1 2, 50 2/10 1-2/50
ON_Eth Al-CB 1<1 1<1 0/0 1<1* 1/1 0 3, 100 1/5 0/0
ON_For Al-CB 3/90 3/90 3, 90 3/100 3/50 3/100
ON_Cyt Al-CB 0/* 1<1 0/0 1/* 2/10 2/50 1-3/10 1<1
SD_Cyt_4-6h Al-CB 1<1 1/10
SDy_For Cytospn 3 3 3/100
SDy_Cyt Cytospn n2, c1 3 3, 100 3/100
ON_Cyt Cytospn no post fix 1-2/30 3/100
ON_Cyt Cytospn form_post 1, 10
ON_Cyt Cytospn acet_post 2/30
SDy_Eth<5” Al-CB 1<1 3 2/5
SDy_Eth<30” Al-CB 1<1
SDy_For30” Al-CB 3/100 3/100 3/70 3 >50
SDy_For60” Al-CB 3/100 3/100 3/70
SDy_For120” Al-CB 3/100 3/100 3/70
Estrogen Receptor SP1 * = Rare cells
ER-Serous Ca; Albumin; FT08 Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
ER-Serous Ca; Albumin; FT09 Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
ER- Alcohol and Formalin Serous carcinoma FT19
FT008 FT009 FT010 FT013 FT 019 FT020 FT021 FT025 FT027 FT 33 FT 34 FT 35 FT 41 FT43 FT45 FT48
Ser Ser Renal Ser Ser Ser Ser Renal Ser Renal Ser Ser Ser Ser Ser Renal
Fixative Prep PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX8 PAX PAX PAX-8
SD_Eth Al-CB 3 2/10 1<1 3/20 3/100 3,/75 3/90
SD_For Al-CB 3 3/100 3/10 3/100 3/100 3/100 3/90 3/90 3/100 3/100 3/100 3/100
SD_Cyt Al-CB 2 2/90 1<1 2/100 3/70 2/90 1-2, 30 2/10 3/100 3/25 3/80
ON_Eth Al-CB 1/5 1/5 1/50 2/20 2/100 2/50* 2/1 2/80 2/10 3/100 2,50 1-3/80
ON_For Al-CB 3/50 3/100 3/100 3/100 3/100 3/100 3/100
ON_Cyt Al-CB 1-2/80 2/* 1/1 1-2/80 2/50 3/100 3/100 1-3/ 50 2-3/80 1-2/80
SD_Cyt_4-6h Al-CB 1-3/80
SD_For Cspn 3/100 3/100 3/100
SD_Cyt Cspn 3/80 3/100
ON_Cyt Cspn 3/90 3/90 1-3/100
ON_Cyt Cspn form_post 3/90
ON_Cyt Cspn acet_post 3/90
no prefix Al-CB 3/100 3/100 2/10
SD_For30' Al-CB 3/100 3/100 3/90 3/100 3/90
SD_For60' Al-CB 3/100 3/100 3/90
SD_For120' Al-CB 3/100 3/90
PAX-8 * = Rare cells
PAX8 FT25; Renal Cell Carcinoma:
FT41; Serous Carcinoma:
Fixative PREP FT08 FT09 FT 19 FT20 FT21 FT34 FT35 FT41 FT43 FT45
WT1 WT1 WT1 WT1 WT1 WT1 WT1 WT1 WT1 WT1
SD_Eth Al-CB 1-3/50 3/100 3/100 3/100 3/1
SD_For Al-CB 2-3/60 3/100 3/100 3/100 3/90 3/100 3/100 3/1 3/100
SD_Cyt Al-CB 1-2<10 3/100 1/90 3/70 3/100 3/75 0
ON_Eth Al-CB 1<1 2-3/90 2/100 3/100 2/30 3/100 2/75 0
ON_For Al-CB 2-3/50 3/100 3/80* 3/100 3/100 3<1 3/100 3/100
ON_Cyt Al-CB 1-2/20 2-3/90 1-2/80 3/* 2/50 3/100 3/100 0 2-3/90
SD_Cyt_4-6h Al-CB 0
SD_For Cspn 2*
SD_Cyt Cspn 3/1
ON_Cyt Cspn 3/1
no prefix Al-CB 3/100 3/100 3/30
SD_For30' Al-CB 3/100 3/100 3/90
SD_For60' Al-CB 3/100 3/100 3/90
SD_For120' Al-CB 3/100 3/90
WT-1 * = Rare cells
WT1 Serous Ca FT8 Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
WT1 Serous Ca FT09
Same day - preserved within the hour; processed after one hour
Overnight – preserved within the hour; processed the next day
TTF-1 (Lung) Fixative PREP FT 36 FT 36 FT 36 FT 38 FT 39 FT 40
TTF Nap p63 TTF TTF TTF
SD_Eth Al-CB 3, 100% 3, 100% 0 3, 100% 3, 100% 3, 100%
SD_For Al-CB 3, 100% 3, 100% 0 3, 100% 3, 100% 3, 100%
SD_Cyt Al-CB 3, 100% 2, 100% 0 3, 100% 3, 100% 3, 100%
ON_Eth Al-CB 3, 100% 3, 100% 0 3, 100% 3, 100% 3, 100%
ON_For Al-CB 3, 100% 3, 100% 0 3, 100% 3, 100% 3, 100%
ON_Cyt Al-CB 3, 100% 3, 100% 0 3, 100% 3, 100% 2, 100%
SD_For Cspn 3, 100% 2, 100% 0 3, 100% 3, 100% 3, 100%
SD_Cyt Cspn 3, 100% 3, 100% 0 3, 100% 3, 100% 3, 100%
ON_Cyt Cspn 3, 100% 2, 100% 0 3, 100% 3, 100% 3, 100%
TTF-1 Lung Adenoca FT36
Napsin A Lung Adenoca FT36
Fixative PREP
FT10 FT10 FT25 FT25 FT48 FT48 FT48 FT48 FT48 FT48
CD10 EMA CD10 EMA EMA CAIX Vimentin PAX-8 MOC31 BerEp4
SD_Eth Al-CB 1<1 2/10
SD_For Al-CB 3<10 0/0 3/100
SD_Cyt Al-CB 1<5 1/5 0/* 3/100
ON_Eth Al-CB
ON_For Al-CB
3/100 3/100 3/100 3/100 3/60 2/60
ON_Cyt Al-CB
3/100 1-2/90 3/100 1-2/80 2/80 1/30
RENAL CELL CARCINOMAS
* = Rare cells
Renal Cell Carcinomas Renal Cell Carcinoma FT48
Renal Cell Carcinoma FT25
Quality matters Pleural Fluid – Lung Mets for EGFR
ANTIBODY Cases ETHANOL FORMALIN CYTOLYT VENDOR CLONE DIL
EPITHELIAL Ber-EP4 7 good good good Dako Canada Inc Ber-EP4 1:25
MOC-31 9 good good good Dako Canada Inc MOC-31 1:50
MESOTHELIAL Calretinin 6 variable good variable Ventana SP65 Pre-diluted
CK5/6 4 good good good Dako Canada Inc D5/16B4 1:50
D2-40 4 variable good good Dako Canada Inc D2-40 1:100
DIFFERENTIAL WT1 10 variable good variable Leica - Novocastra WT49 1:10
ER 13 variable good variable Ventana SP1 Pre-diluted
PAX8 16 variable good variable Cedarlane/ProteinTech Group Inc Polyclonal 1:250
TTF-1 4 good good good Leica - Novocastra SPT24 1:100
Napsin A 1 good good good Leica - Novocastra IP64 1:200
EMA 3 good good Dako Canada Inc E29 1:25
CDX2 2 good good good Dako Canada Inc DAK-CDX2 1:20
CK7 1 good good Dako Canada Inc OV-TL 12/30 1:50
CK20 1 good good Dako Canada Inc Ks20.8 1:50
Mammaglobin 1 good good Dako Canada Inc 304-1A5 1:100
GCDFP-15 1 no stain no stain Dako Canada Inc 23A3 1:50
S100 1 good good Dako Canada Inc Polyclonal 1:3000
Melan-A 1 good good Dako Canada Inc A103 1:25
HMB45 1 good good Dako Canada Inc HMB45 1:100
p53 1 good good Dako Canada Inc DO-7 1:100
Summary of stains
Nuclear stains
• Caution with negative ER on alcohol fixed material
• PAX-8 and WT-1 generally reliable in all fixation settings
• TTF1 was reliable in all fixation settings
Cytoplasmic stains
• Epithelial: MOC31 and BerEp4 were reliable in all settings
• Other stains appear reliable but numbers too small
Mesothelial stains
• CK5/6 and Calretinin (cytoplasmic) were positive in some carcinomas
• D2-40 appears to be more specific for mesothelial cells
Summary - Specific
Summary - General
• Formalin preservation is best for immunostains.
• Alcohol preservation gives better morphology but may be unpredictable with some immunostains
• Alcohol - short time exposure usually not a problem but prolonged exposure leads to less staining.
• Alcohol preservation overnight expands the cell button volume and gives appearance of fewer cells
• ‘Mesothelial’ cells are often histiocytes.
• Take Home: Pre analytic variables matter and appropriate controls are important.
Acknowledgments Patients who consented to give fluids for this study Vancouver Cancer Center Staff Amir Rahemtulla, MD: Medical Director, Dept of Pathology and Lab Medicine
Cytology laboratory technologists Ron Garbuio (section head)
Zelma Edgar (ret’d)
Paul Johnson
Anita Chiu
Eric Siksik
Gillian Theed (clerical)
Histology laboratory Nadia Gale (section head)
Chris Dawe
Yin Ping Lee
School of cytology Brenda Smith (Teaching technologist)
Medical Day care Nancy Runzer and staff
Questions
• Tissue controls are reliable for assessing immunostains on cytology cell blocks.
a. T
b. F
• Immunohistochemical stain sensitivity is generally highest with which cell preservative:
a. methanol
b. ethanol
c. formalin
