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

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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