søren nielsen ihc pitfalls - leica 2011
TRANSCRIPT
Generel pitfalls in diagnostic IHC
Søren NielsenScheme ManagerNordiQCAalborg Hospital, Denmark
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IHC quality consultant at Institute of Pathology, Aalborg, Denmark
> 55.000 IHC slides annually 6 BenchMark Ultra, Ventana 1 Autostainer Link 48, Dako 1 Celerus Wave, Celerus Diagnostics 1 Leica Bond III
IHC cooperation partners Roche / Ventana Cell Marque Dako Thermo Fischer Leica
IHC – Most common pitfalls
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IHC - Most common pifalls
www.nordiqc.o
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IHC - Most common pifalls
DecalcificationPreparation Pre-
analytic
Analytic
Post-analytic
TissueType, Dimension,Laser resection,De-differentiation
FixationTime, Type, Volume
SectionThicknessStorageDrying Visualization
Sensitivity, Specificity
Primary antibodyClone, Dilution Buffer, Time, Temp
DevelopmentSensitivity,Localization
InterpretationLocalizationPositive/Negative - cut-off level
QuantificationReporting
Controlment
Pre-treatment
The IHC multi-parameter
complexity
ManualStainer
IHC – Most common pitfalls
DecalcificationPreparation Pre-
analytic
Analytic
Post-analytic
TissueType, Dimension,Laser resection,De-differentiation
FixationTime, Type, Volume
SectionThicknessStorageDrying Visualization
Sensitivity, Specificity
Primary antibodyClone, Dilution Buffer, Time, Temp
DevelopmentSensitivity,Localization
InterpretationLocalizationPositive/Negative - cut-off level
QuantificationReporting
Controlment
Pre-treatment
With 3 choices for 5 variables in each phase = >
4 million protocols….
ManualStainer
IHC – Most common pitfalls
Appropriate tissue fixation and processing
Appropriate and efficient epitope retrieval
Appropriate choice of antibody/clone
Robust, specific & sensitive detection system
Appropriate choice of control material
The basal fundament for a technical
optimal IHC performance:
IHC – Most common pitfalls
Appropriate tissue fixation and processing– Problem 1: Too short fixation in NBF– Problem 2: Delayed fixation in NBF– Too long fixation in NBF is not a
problem !!!
IHC – Most common pitfalls
Appropriate tissue fixation and processing– Problem 1: Agressive decalcification – Problem 2: Deviation from SOP – e.g. section
baking
False negative or false positive
6 - 48h
8 - 72h
IHC – Most common pitfalls
Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma.
Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F
Am J Clin Pathol. 2003 Jul;120(1):86-92
“ The minimum formalin fixation time forreliable immunohistochemical ER results is 6 to 8 hours in our laboratory, regardless of the type or size of specimen(core biopsy or resection)”.
IHC – Most common pitfalls
IHC – Most common pitfalls
13 hours versus 79 hours in 10% NBF (the week-end dilemma…..)
101 breast carcinomas:
99 % Concordance between short fixation and long fixation for ER (SP1)
95 % Concordance between short fixation and long fixation for PR (1E2)
98 % Concordance between short fixation and long fixation for HER2 (A0485)
IHC – Most common pitfalls
PR (5-10%) PR (30%)
SF score 3 PF score 6
IHC – Most common pitfalls
InternalIHC validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 + + + +Tumour 2 3+ + + + +Tumour 3 + + + +Tumour 4 + + + +Tumour 5 + + + +Tumour 6 3+ + + + +Tumour 7 + + + +Tumour 8 + + + +Tumour 9 + + + +
Breast carcinomas, HER-2 PATHWAY, rmAb 4B5
(CC1 Mild, Ab inc. 20 min. 36°C, UltraView DAB)
IHC – Most common pitfalls
4 h
48 h
24 h
168 h
Breast carcinoma 3+, HER-2 PATHWAY, rmAb 4B5
IHC – Most common pitfalls
4 h
48 h
24 h
168 h
Breast carcinoma 1+, HER-2 PATHWAY, rmAb 4B5
IHC – Most common pitfalls
InternalSISH validation
4 h. NBF 24 h. NBF 48 h. NBF 168 h. NBF
Tumour 1 + + + -Tumour 2 Amp + + + +Tumour 3 (+) + - -Tumour 4 + + - -Tumour 5 + + + +Tumour 6 Amp + + + +Tumour 7 - + + -Tumour 8 poly. + + + -Tumour 9 poly. + + + -
HER-2 ISH:
Breast carcinomas, Dual SISH CCrb ext, P3. 8 m
IHC – Most common pitfalls
4 h
48 h
24 h
168 h
Breast carcinoma, 1+ Dual SISH CCrb ext, P3. 8 m
IHC – Most common pitfalls
”0” day Fixation
Fixed in toto 4 hours
- sliced and processed
IHC – Most common pitfalls
IHC – Most common pitfalls
30 min delay
a: HER2 IHC
b: HER2 ISH
2 hours delay
c: HER2 IHC
d: HER2 ISH
CD4 – SP35
IHC – Most common pitfalls
IHC – Most common pitfalls
CD10 – 56C6
IHC – Most common pitfalls
Prostate – Ki67, rmAb clone 30.9
10 % NBF 24h → 24h 10 % form. acid 10 % NBF + 10 % form. acid 24h
IHC – Most common pitfalls
Prostate – PIN cocktail – p504s/CK5/p63
10 % NBF 24h → 24h 10 % form. acid 10 % NBF + 10 % form. acid 24h
60°C 1h. HER-2: 3+ 80°C 16h. HER-2: 1+
IHC – Most common pitfalls
Appropriate tissue fixation and processing
Appropriate and efficient epitope retrieval
Appropriate choice of antibody/clone
Robust, specific & sensitive detection system
Appropriate choice of control material
The basal fundament for a technical
optimal IHC performance:
IHC – Most common pitfalls
Appropriate and efficient epitope retrieval
– Problem 1: Too short effcient HIER period
– Problem 2: Use of a non-alkaline HIER buffer
– Problem 3: Wrong epitope retrieval type
IHC – Most common pitfalls
False negative or false positive
Pre-treatment / Epitope retrieval:
Defined as an unmasking method
for ”re-storing” blocked antigens
in formaldehyde fixed tissue
The key to an optimal IHC reaction…
IHC – Most common pitfalls
Optimized temperature-time-pH-buffer system
‘Heating condition’ = temperature × time:
121ºC/1 min 100ºC/20 min 95ºC/40 min 60ºC/24 h.
Heat Induced Epitope Retrieval
Device:Water bathMWOPressure cookerPressure cooker & MWOAutoclaveSteam
Considerations:EfficiencyStandardizationTissue damagePerformance
IHC – Most common pitfalls
Domestic Prestige Polar Patent Pascal
Milestone
Ref. A.J. Balaton et al. Appl. Immunohistochem. 4(4):259-263,1996
MWO Pressure cooker
10
20
IHC – Most common pitfalls
Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma.
Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F
Am J Clin Pathol. 2003 Jul;120(1):86-92
IHC – Most common pitfalls
HIER CC1
stand. 60 min.
IHC – Most common pitfalls
Ton 168h 10 % NBF
HIER CC1
short 8 min.
Ton 24h 10 % NBFTon 6h 10 % NBFCD23
rmAb SP23
IHC - most common pitfalls
NQC B10
HER-2
HER-2 Optimal Good Borderline Poor
HercepTest™ 38 3 0 9
82 % (41/50) 18 % (9/50)
NordiQC 89 % (134/151) 11 % (17/151)
IHC - most common pitfalls
HercpTest as package insert
Waterbath ?? - all protocols based on PT-Link obtained an sufficient result..(n=19)
IHC – Most common pitfalls
A: CD20, cl. L26
B: Ki67, cl. MIB1
C: HMB45
(D: MOC31)
Modified from: Shi et al. J Histochem Cytochem 1995 43:193-201
6 8 - 9
IHC – Most common pitfalls
IHC – Most common pitfalls
Tonsil
24 h NBF
HIER
Pascal PC
TE pH 9
Ci pH 6
CD3 PS1 CD19 LE-CD19 PMS2 A16-4
10/1 mM Tris/EDTA pH 9 ~
• Target Retrieval Solution pH 9 S2367/2368, Dako
• Cell Conditionning 1 (CC1) pH 8.5, Ventana
• Bond Epitope Retrieval Solution 2, Leica Microsystems
Citrate pH 6 ~
• Target Retrieval Solution Citrate pH 6, S2369, Dako
• Cell Conditionning 2 (CC2) pH 6.0, Ventana (cave detergent….!!)
• Bond Epitope Retrieval Solution 1, Leica Microsystems
IHC – Most common pitfalls
39
TE9 optimum Ci6 optimum
TE9 excessive
IHC – Most common pitfalls
40Optimal Excessive
IHC – Most common pitfalls
CD10
IHC completed – wash, mounted IHC completed – dried before mounted
IHC – Most common pitfalls Ki67 mAb clone MIB1 – HIER CC1 standard BenchMark Ultra
IHC completed – wash, mounted IHC completed – dried before mounted
IHC – Most common pitfalls CD79a rmAb clone SP18 – HIER CC1 standard BenchMark Ultra
IHC – Most common pitfalls
Laminin polyclonal Z0097
HIER ER 2 / TE Proteolysis Pepsin / P1
Appropriate tissue fixation and processing
Appropriate and efficient epitope retrieval
Appropriate choice of antibody/clone
Robust, specific & sensitive detection system
Appropriate choice of control material
The basal fundament for a technical
optimal IHC performance:
IHC – Most common pitfalls
Appropriate choice of Ab / clone
– Problem 1: Provides low sensitivity
– Problem 2: Provides low specificity
– Problem 3: Selective to IHC platform
IHC – Most common pitfalls
False negative or false positive
Optimally test > 1 clone / Ab before implementation
Use instructions from vendor as guideline (class 1 reagents)
Compare with observations from CiQC, NordiQC, UK-NEQAS, litterature and colleages
Validate on own processed material with own IHC platform
Why not just use instructions from vendor ??
IHC – Most common pitfalls
TonsilPLAP antibody
Clone PL8-F6 - Internal validation and protocol set-up
IHC – Most common pitfalls
Tonsil User ProtocolBioGenex RecommendationsParameterSuper Sensitive/Concentrated
ParaffinTissue TypeMWO/TENonePretreatment
Placenta or Germ cell neoplasmControl Tissue5050 - 100Concentrated Dilution30 min., room temp.30 min., room temp.Incubation Time & Temp.
10 - 12 mg/mlProtein ConcentrationAM2280200SLot No:
Prod.
PLAP antibody
Clone PL8-F6 - Internal validation and protocol set-up
IHC – Most common pitfalls
MB Ab test 1
clone PL8-F6
1:50 Recommendation 1:50 Own optim. + HIER
IHC – Most common pitfalls
Placenta
Seminoma
1:25 / 100 (mo/po) 1:100 / 400 1:400 / 2000
A None None NoneB Enzyme 1, 8 min Enzyme 1, 8 min Enzyme 1, 8 minC HIER ER 2 pH 9.0 HIER ER 2 pH 9.0 HIER ER 2 pH 9.0 D HIER ER 1 pH 6.0 HIER ER 1 pH 6.0 HIER ER 1 pH 6.0
(E) ER 1 + Enzyme 1, 4 min ER 1 + Enzyme 1, 4 min ER 1 + Enzyme 1, 4 min
(F) Enzyme 1, 4 min + ER 1 Enzyme 1, 4 min + ER 1 Enzyme 1, 4 min + ER 1
Same detection system - sensitive, specific, universal (Refine)Only variable in the laboratory is then the pre-treatment !
Test specimens with different levels of the antigen (none, low, medium, high)Optimally compare more clones before implementation
IHC – Most common pitfalls
Ton 4h Ton 24h Ton 72h Ton 168h
Colon Liver Kidney Pancreas
Placenta Lung Prostate Breast
Skin Brain
MB Ab test 1
IHC – Most common pitfalls
TongueTon 24h +
decalc.
Tumor 1 Tumor 2 Tumor 3 Tumor 4
Tumor 5 Tumor 6 Tumor 7 Tumor 8
Tumor 9 Tumor 10 Tumor 11 Tumor 11
Tumor 12 Tumor 13 MB Ab test 2
IHC – Most common pitfallsTMA Neoplasia
Liver
Mamma ductal carc.
Mamma ductal carc.
Mamma Lobular
carc.
Lung adeno carc.
Lung adeno carc.
Lung squam.
carc.
Colon adeno carc.
Colon adeno carc.
Kidney clear c carc.
Kidney clear c carc.
Thyroid. follic. carc.
Thyroid. Medul. carc.
Ovary. Serous I
carc.
Ovary. Serous I
carc.
Ovary. Clear carc.
Ovary. Endom.
carc.
Corpus Uteri
Endom. carc.
Cerxix Uteri adeno carc.
Testis Semin.
Testis Semin.
Tonsil Prostateaadeno carc.
Prostate adeno carc.
Intest Carcinoid
Melanom Melanom Pancr. adeno carc.
Pancr. adeno carc.
Uroth. carc.
Uroth. carc.
Hodgkin Classic
Mantle cell
lymph.
Hodgkin mixed
Diffuse large B lymph.
Diffuse large B lymph.
Follik. lymph
B-CLL T-cell lymph. perip.
T-cell lymph. Anapl.
GIST Leio myo
sarcoma
Rhabdomyo
sarcoma
MLH1 clone ES05, 1:20 (Ref.) MLH1 clone EPR3894, 1:250
IHC – Most common pitfalls MLH1 test – Tonsil fixed 4 h NBF
MLH1 clone ES05, 1:20 (Ref.) MLH1 clone EPR3894, 1:250
IHC – Most common pitfalls MLH1 test – Colon ad. carc. 1, MLH1 negative – DNA mutation (PCR)
CD138 clone MI15, 1:100 TE CD138 clone 5F7, 1:50 TE
IHC – Most common pitfalls CD138 test – Tonsil fixed 24 h NBF
CD138 clone MI15, 1:100 TE CD138 clone 5F7, 1:50 TE
IHC – Most common pitfalls CD138 test – Plasma cytoma fixed 24 h NBF
Ref: rmAb EPR2764Y CM 1:2532M/CC1S/UV+AMP
rmAb SP54 1:10016M/CC1M/UV
IHC – Most common pitfalls CDX2 test
Ref: rmAb EPR2764Y CM 1:2532M/CC1S/UV+AMP
rmAb SP54 1:10016M/CC1M/UV
IHC – Most common pitfalls CDX2 test
Ref: rmAb EPR2764Y CM 1:2532M/CC1S/UV+AMP
rmAb SP54 1:10016M/CC1M/UV
IHC – Most common pitfalls CDX2 test
Ref: rmAb EPR2764Y CM 1:2532M/CC1S/UV+AMP
rmAb SP54 1:10016M/CC1M/UV
IHC – Most common pitfalls CDX2 test
Ref: rmAb EPR2764Y CM 1:2532M/CC1S/UV+AMP
rmAb SP54 1:10016M/CC1M/UV
IHC – Most common pitfalls CDX2 test
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IHC – Most common pitfallsAntigen Clone High
expressorLow
expressorNon
expressor
CD5 CD5/54/F6 √ FN –
CD23 MHM6 √ FN –
CD31 1A10 (√) FN –
CD31 SP54 (√) FN FN, FP
CD138 5F7 (√) FN –
CEA TF-3H8-1 √ √ FP
CGA DAK A3 √ FN –
CK-LMW CAM 5.2 √ (√) –
MLH1 EPR3894 √ √ FP
MSH2 EPR3943 √ √ FP
PR SP2 √ √ FP
SYP SY38 √ FN –
TTF1 8G7G4/1 √ FN FP
63
IHC – Most common pitfallsAntigen Clone XT / Ultra Autostainer Bond-max
CD4 1F6, 4B12 FN (3%H2O2) √ √
CD4 SP35 √ √ √
CD5 4C7 FP √ √
CD5 SP19 √ √ √
CD79a JCB117 Weak √ √
CD79a SP18 √ √ √
ASMA 1A4 (√) Weak √ √
BSAP 24 FN √ (- Weak)
BSAP SP34 √ √ √
BCL6 PG-B6p FN (3%H2O2) √ √
BCL6 GI191E/A8 √ √ √
Oct-2 OCT-207 FN √ ?
Oct-2 MRQ-2 √ ? ?“IHC-Platform” depending markers”
IHC – Most common pitfalls
mAb clone 123C3:
Non-VMS 81% passed
VMS 13% passed
rmAb clone MRQ-42
VMS 100% passed
IHC – Most common pitfalls
mAb clone A103:
Bond 75% passed
VMS 12% passed
IHC – Most common pitfalls
1:100, PC TE pH 9, EnV+, AS+ 1:100, CC1 pH 8.5, Ul.W., Ultra
IHC – Most common pitfalls SF-1 test – mAb clone N1665 R&D systems
Appropriate tissue fixation and processing
Appropriate and efficient epitope retrieval
Appropriate choice of antibody/clone
Robust, specific & sensitive detection system
Appropriate choice of control material
The basal fundament for a technical
optimal IHC performance:
IHC – Most common pitfalls
Robust, specific & sensitive detection system
– Problem 1: Use of biotin based detection
systems
– Problem 2: Use of detection systems with
low sensitivity
IHC – Most common pitfalls
False negative or false positive
IHC – Most common pitfalls
Biotin based detection systems should not be used for IHC….!
EnVision Flex / Flex +
UltraView
UltraVision LP
UltraVision One
Refine
ImPress
Super Sensitive
Super Picture
Hi Def
Quanto
Advance
MACH 3
.......
IHC – Most common pitfalls
Sensitivity
PriceTAT
The choice of a polymer / multimer based system
Quanto
Hi Def.
EnV.Fl.+
Refine
Super Sens.
Ultra Vis. LP UltraView+amp
Ult.Vis. ONE EnV. Fl.
UltraView
Sensitivity
1:25 1:50 1:150 1:500
Com
plex
ityIHC – Most common pitfalls
Indirect method: 2-step polymer method
EnVision+, Env. FlexUltraViewUltraVision-one
IHC – Most common pitfalls
Traditionel 3-step polymer based detection system:
Ultravision LP, Refine, Super Sensitive, PowerVision+, Novolink – amp. mouse ab
Second generation 3-step polymer based detection systems:
EnVision Flex+, UltraView + amplification, Quanto, Hi-Def – amp. mouse & rabbit ab
Mouse ab.
Rabbit anti-mouse
Polymer goat anti-rabbit
Polymer goat anti-mouse
1. Primary ab.
2. Link ab.
3. Polymer cocktail
IHC – Most common pitfalls
IHC – Most common pitfalls
PAX5 SP34 RTU – 32 min in primary, HIER CC1 standard:
3-step Multimer system (UltraView + Amplification) Tonsil & Hodgkin
2-step Multimer system (UltraView)
IHC – Most common pitfalls
IHC – Most common pitfalls
IHC – Most common pitfalls
HIER alkaline buffer + 3-step polymer: 89% sufficient, 67% optimal
HIER alkaline buffer + 2-step polymer: 32% sufficient, 13% optimal
VMS XT/Ultra IHC platform: 24% suffcient, 0% optimal
BOND IHC platform: 77% suffcient, 62% optimal
IHC – Most common pitfalls
Appropriate tissue fixation and processing
Appropriate and efficient epitope retrieval
Appropriate choice of antibody/clone
Robust, specific & sensitive detection system
Appropriate choice of control material
The basal fundament for a technical
optimal IHC performance:
IHC – Most common pitfalls
Appropriate choice of control material– What normal tissue ?
– Non-, low-, medium and high-expressor
– Processed as diagnostic tissue
IHC – Most common pitfalls
Begin at the beginning,' the King said gravely, `and go on till you come to the end: then stop.'
Alice in Wonderland
For IHC: begin at the end, tune in your protocol: then stop.
MB K1 APPENDIX TONSIL PANCREAS HEPARASMAAlfa-smooth muscle actin(Cytopl)
Smooth muscle cells in vessels and in muscle layers. Myofibroblasts lining the epithelial surface
Smooth muscle cells in vessels
Smooth muscle cells in vessels
Smooth muscle cells in the liver sinusoids
B-CATENINBeta-catenin(Membrane)
Membranes of columnar epithelial cells. Endothelial and follicular denritic cells
Membranes of squamous epithelial cells
Membranes of acinar epithelial cells (ducts) and endocrine cells
Hepatocytes - weak membranous
BCL2Bcl2-oncoprotein(Cytopl)
A weak to moderate staining in the epithelial cells in the basal crypts
All peripheral lympho-cytes incl T-cells in germinal centres - Germinal center B-cells are negative.
Weak reaction of epithelial cells.
Weak reaction of the epithelial cells in bile ducts.
BCL6Bcl6-protein(Nuclear)
Germinal center B-cells Germinal center B-cells and basal squamous cells ? ?
CD2(Membrane)
All T-cells - Scattered intraepithelal T-cells
All T-cells - Scattered T-cells in germinal centres
T-cells T-cells
CD3(Membrane)
All T-cells - Scattered intraepithelal T-cells
All T-cells - Scattered T-cells in germinal centres
T-cells T-cells
CD4(Membrane)
60 - 80 % of T-cells 60 - 80 % of T-cells and Germinal center macrophages
T-cells Kupfercells and sinusoid endothelial cells
CD5(Membrane)
All T-cells - Scattered intraepithelal T-cells
All T-cells - Scattered B-cells in the mantle zone must show a weak membranous staining
T-cells T-cells
CSQI
Critical
Staining
Quality
Indicator
IHC – Most common pitfalls
CD23
CSQI:
Activated B-cells in mantle z.
IHC – Most common pitfalls
CDX2
CSQI:
Pancreatic duct ep. cells
IHC – Most common pitfalls
85
NordiQC consensus marks
Optimal: 36 % Good: 33 % Borderline: Poor:
too weak / false neg.: ~ 90 %
over-stained / false pos.: ~ 10 %{31 %}
IHC – Most common pitfalls
86
Major causes of insufficient stains:
Less successful / problematic antibodies 18 %
Inappropriate antibody dilution 39 %
Inappropriate epitope retrieval 31 %
Other inappropriate lab. performance 12 % Inappropriate calibration / home brews Endogenous biotin reaction (EBR) Section drying-out after HIER Technical stainer errors . . . . Unexplained
IHC – Most common pitfalls
“Leading”
IHC – Most common pitfalls
IHC is a challenge, technical complex but not mission impossible and rests on 5 legs
Use proper controls - normal tissue! Use a robust and specific detection system Use efficient HIER Use Ab clones, optimal for the IHC platform Harmonize and standardize tissue processing