IHC Technology – Problems Solutions

IHC Technology Problems and Solutions Lecture Workshop Presented By Lawrence Richards M.S.,H.T(ASCP).,QIHC(ASCP)

Introduction Quality in IHC starts at the Surgical Room Immunohistochemistry moved from being a Stain into an Immuno Assay. Strict GLP protocol and QC is very important for a reliable, sensitive, specific, reproducible results.

Quality in IHC starts at the

Surgical Room

Immunohistochemistry moved from being a Stain into an Immuno Assay.

Strict GLP protocol and QC is very important for a reliable, sensitive, specific, reproducible results.

Preanalytical variables Pre-Analytical Variables: Surgical Room Pathology Lab IHC Lab 1. Surgical Room Method of tissue removal Time between surgery and Fixation

Pre-Analytical Variables:

Surgical Room

Pathology Lab

IHC Lab

1. Surgical Room

Method of tissue removal

Time between surgery and Fixation

Preanalytical variables contd Pathology Lab Grossing Fixatives Storage

Pathology Lab

Grossing

Fixatives

Storage

Preanalytical variables contd IHC Lab Receiving Processing Microtomy Microscope slides Drying slides Storage of cut slides

IHC Lab

Receiving

Processing

Microtomy

Microscope slides

Drying slides

Storage of cut slides

Other IHC variables Room Temperature : + or – 10*C can have a difference between 2+ or 3+ staining Heat : 37*C affects ER/PR as much as 10 fold reduction. Other heat labile Ab:ALK1,CD3,CD4,CD10,CD43,CD45RO, CyclinD1,ER,PR,MIB1,TTF-1,TdT Agitation : Most improved with agitation, watch out for low affinity Ab eg ER(1D5) Washing :Can dislodge low affinity binding, Long washing in tap water causes background

Room Temperature : + or – 10*C can have a difference between 2+ or 3+ staining

Heat : 37*C affects ER/PR as much as 10 fold reduction. Other heat labile Ab:ALK1,CD3,CD4,CD10,CD43,CD45RO,

CyclinD1,ER,PR,MIB1,TTF-1,TdT

Agitation : Most improved with agitation, watch out for low affinity Ab eg ER(1D5)

Washing :Can dislodge low affinity binding, Long washing in tap water causes background

ER Sub-optimal staining Cervix NordiQC Insufficient HIER Clone not robust 1 0 / 2 0 Too low conc Epitopes lost

Insufficient HIER

Clone not robust

1 0 / 2 0 Too low conc

Epitopes lost

Polymer Based 2 Step:ENVISION DAKO

Labelled Streptavidin-Biotin(LSAB) DAKO

IHC Assay Pre-Treatment Blocking Primary Antibody Detection System Chromogen Counter Stain

Pre-Treatment

Blocking

Primary Antibody

Detection System

Chromogen

Counter Stain

Pre-treatments Epitope Unmasking Two types : Enzyme and HIER . Enzyme : Trypsin, Pepsin, Pronase, Ficin, Prot K, Protease 24, Saponin. HIER: Types: WB, Pressure- cooker,Microwave,Autoclave….. Buffers: NaCit, EDTA,Citric Acid, Buffers… Why not both? Enzyme & HIER .

Two types : Enzyme and HIER .

Enzyme : Trypsin, Pepsin, Pronase, Ficin,

Prot K, Protease 24, Saponin.

HIER:

Types: WB, Pressure- cooker,Microwave,Autoclave…..

Buffers: NaCit, EDTA,Citric Acid, Buffers…

Why not both? Enzyme & HIER .

Pre-Treatment contd Cit Buff pH6 Tris-EDTApH9 Autoclave 121*C 10’ WB 95*C 40’ MW 5’ X 3 MW 10’ X 3 Source:Hasegawa,DAKO-Connection 2008

ER @ pH6 ER @ 9.5pH

Antigen Retrieval Figure 1. Comparison of intensity of AR-IHC by using the test battery for MAb MIB1 on sections of tonsil ( A-J ) and MAb to thrombospondin on sections of bladder carcinoma ( K-T ). The AR protocols used are arranged in the following order: pH 1, 100C, 20 min (A,K); pH 1, 100C, 10 min (B,L ); pH 1, 90C, 10 min ( C,M ); pH 6, 100C, 20 min ( D,N ); pH 6, 100C, 10 min ( E,O ); pH 6, 90C, 10 min ( F,P ); pH 10, 100C, 20 min (G,Q ); pH 10, 100C, 10 min ( H,R ); pH 10, 90C, 10 min ( I,S ); no AR pretreatment ( J,T ). For MIB 1, the strongest intensity of staining was found in A, B, and G . However, pH 1 showed a low background level of false nuclear staining, and the hematoxylin stain did not take well compared to pH 10, 100C, 20 min (G). Not shown :citrate buffer of pH 6 yield a stronger staining of MIB1 than Tris-HCl, pH 6 as per G G A B Shan-Rong Shi a , Richard J. Cote a , and Clive R. Taylor a Journal of Histochemistry and Cytochemistry, Vol. 45, 327-344

Figure 1. Comparison of intensity of AR-IHC by using the test battery for MAb MIB1 on sections of tonsil ( A-J ) and MAb to thrombospondin on sections of bladder carcinoma ( K-T ). The AR protocols used are arranged in the following order:

pH 1, 100C, 20 min (A,K);

pH 1, 100C, 10 min (B,L );

pH 1, 90C, 10 min ( C,M );

pH 6, 100C, 20 min ( D,N );

pH 6, 100C, 10 min ( E,O );

pH 6, 90C, 10 min ( F,P );

pH 10, 100C, 20 min (G,Q );

pH 10, 100C, 10 min ( H,R );

pH 10, 90C, 10 min ( I,S );

no AR pretreatment ( J,T ).

For MIB 1, the strongest intensity of staining was found in A, B, and G .

However, pH 1 showed a low background level of false nuclear staining, and the hematoxylin stain did not take well compared to pH 10, 100C, 20 min (G).

Not shown :citrate buffer of pH 6 yield a stronger staining of MIB1 than Tris-HCl, pH 6 as per

Antigen Retrieval Figure 1. Comparison of intensity of AR-IHC by using the test battery for MAb MIB1 on sections of tonsil ( A-J ) and MAb to thrombospondin on sections of bladder carcinoma ( K-T ). The AR protocols used are arranged in the following order: pH 1, 100C, 20 min ( A,K ); pH 1, 100C, 10 min ( B,L ); pH 1, 90C, 10 min ( C,M ); pH 6, 100C, 20 min ( D,N ); pH 6, 100C, 10 min ( E,O ); pH 6, 90C, 10 min ( F,P ); pH 10, 100C, 20 min ( G,Q ); pH 10, 100C, 10 min ( H,R ); pH 10, 90C, 10 min ( I,S ); no AR pretreatment ( J,T ). For MIB 1, the strongest intensity of staining was found in A, B, and G . However, pH 1 showed a low background level of false nuclear staining, and the hematoxylin stain did not take well compared to pH 10, 100C, 20 min ( G ). Not shown :Citrate buffer of pH 6 yielded a stronger staining of MIB1 than Tris-HCl, pH 6, as previously reported by Shan-Rong Shi a , Richard J. Cote a , and Clive R. Taylor a Journal of Histochemistry and Cytochemistry, Vol. 45, 327-344

Figure 1. Comparison of intensity of AR-IHC by using the test battery for MAb MIB1 on sections of tonsil ( A-J ) and MAb to thrombospondin on sections of bladder carcinoma ( K-T ). The AR protocols used are arranged in the following order: pH 1, 100C, 20 min ( A,K ); pH 1, 100C, 10 min ( B,L ); pH 1, 90C, 10 min ( C,M ); pH 6, 100C, 20 min ( D,N ); pH 6, 100C, 10 min ( E,O ); pH 6, 90C, 10 min ( F,P ); pH 10, 100C, 20 min ( G,Q ); pH 10, 100C, 10 min ( H,R ); pH 10, 90C, 10 min ( I,S ); no AR pretreatment ( J,T ). For MIB 1, the strongest intensity of staining was found in A, B, and G . However, pH 1 showed a low background level of false nuclear staining, and the hematoxylin stain did not take well compared to pH 10, 100C, 20 min ( G ). Not shown :Citrate buffer of pH 6 yielded a stronger staining of MIB1 than Tris-HCl, pH 6, as previously reported by

Shan-Rong Shi a , Richard J. Cote a , and Clive R. Taylor a Journal of Histochemistry and Cytochemistry, Vol. 45, 327-344

Blocking Endogenous Peroxidase Endogenous Phosphatase Avidin, Biotin Serum Block Protein block

Endogenous Peroxidase

Endogenous Phosphatase

Avidin, Biotin

Serum Block

Protein block

Primary Antibody Selection of Ab or Panel of Abs Types of Ab Polyclonal Vs Monoclonal Rabbit Vs Mouse Monoclonal Titration / Time assay: Checker-board Ab and/or Secondary Conc and Time Temperature: RT, 37*C, 4*C

Selection of Ab or Panel of Abs

Types of Ab

Polyclonal Vs Monoclonal

Rabbit Vs Mouse Monoclonal

Titration / Time assay: Checker-board

Ab and/or Secondary

Conc and Time

Temperature: RT, 37*C, 4*C

Ab Clone Cytokeratin,Low Mol.Wt (CK-LMW) Liver using mAb clone DC10,C51,5D3 or Ks-B17.2 Liver using mAb clone 35 BH11 clone RCC using mAb 35 BH11 RCC using mAb clone DC10,C51,5D3 or Ks-B17.2 NordiQC

Ab problems & solutions Monoclonal vs Polyclonal Diluent Affinity and Bonding Ab reaction time Storage

Monoclonal vs Polyclonal

Diluent

Affinity and Bonding

Ab reaction time

Storage

Controls Poistive and Negative Tissue Controls / Cell Pellets Reagent Control Ab Isotype Controls

Poistive and Negative

Tissue Controls / Cell Pellets

Reagent Control

Ab Isotype Controls

Chromogen (Substrate) Peroxidase AEC DAB Vector S-G Vector-VIP Vector Nova Red Alk Phos New Fuchin Fast Red BCIP/NBT Vector Red Vector Black

Peroxidase

AEC

DAB

Vector S-G

Vector-VIP

Vector Nova Red

Alk Phos

New Fuchin

Fast Red

BCIP/NBT

Vector Red

Vector Black

Counter Stain Hematoxylin Light Green Methyl Green

Hematoxylin

Light Green

Methyl Green

Trouble Shooting No staining Weak Staining Background Tissue falling off Edge Effects Artifacts And million others…..

No staining

Weak Staining

Background

Tissue falling off

Edge Effects

Artifacts

And million others…..

Frequently Used Abs Hospital Labs / Reference Labs Bcl2, CA125, CD3, CD15, CD20, CD30, CD45, CK cocktail, CD7, CK20, Chrom A,Desmin, ER, HER2, HMB45, Ki67, Lysozyme, Melan-A, Mesothelin, PR, PSA, S100, SmMActin, Synaptophysin, TTF-1, Vimentin. CEA, Mammoglobin, WT1, TdT, CMV, Hpylori, HSV1, HSV2, hCG

Hospital Labs / Reference Labs

Bcl2, CA125, CD3, CD15, CD20, CD30, CD45, CK cocktail, CD7, CK20, Chrom A,Desmin, ER, HER2, HMB45, Ki67, Lysozyme,

Melan-A, Mesothelin, PR, PSA, S100, SmMActin, Synaptophysin, TTF-1, Vimentin.

CEA, Mammoglobin, WT1, TdT, CMV, Hpylori, HSV1, HSV2, hCG

Ab Dilution SC  WC = DF DV  DF = A V DV – AV = Diluent Volume SC – Stock conc of Ab µg/ml WC – Working Conc µg/ml DF – Dilution Factor DV – Desired Volume AV – Ab Volume

DV  DF = A V

DV – AV = Diluent Volume

SC – Stock conc of Ab µg/ml

WC – Working Conc µg/ml

DF – Dilution Factor

DV – Desired Volume

AV – Ab Volume

What you need in the IHC Lab Consumables: Ab Detection Systems Chromogen Buffers Antigen Retrieval Reagents

Consumables:

Ab

Detection Systems

Chromogen

Buffers

Antigen Retrieval Reagents

Instruments / Equipments IHC Stainer Antigen Retrieval Water Bath Pressure Cooker Microwave Oven

IHC Stainer

Antigen Retrieval

Water Bath

Pressure Cooker

Microwave Oven

Cost of Starting IHC Lab Place Labour - Training Cost of Equipments Cost of Ab and Reagents Cost of Consumables

Place

Labour - Training

Cost of Equipments

Cost of Ab and Reagents

Cost of Consumables