IHC Technology – ProblemsIHC Technology – Problems

Solutions

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IHC Technology IHC Technology Problems and SolutionsProblems and Solutions

Lecture

WorkshopPresented By

Lawrence RichardsM.S.,H.T(ASCP).,QIHC(ASCP)

IntroductionIntroduction

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.

Pre-clinical Variability Assay Variability

Scoring Variability GLP QC SOP

IHC Assay Development

Preanalytical variablesPreanalytical variables

Pre-Analytical Variables:

1. Surgical Room

2. Pathology Lab / Histology Lab

3. IHC Lab

Surgical Room

Method of tissue removal

Time between surgery and Fixation

Preanalytical variablesPreanalytical variables contdcontd

Pathology Lab

Grossing

Fixatives

Storage

Preanalytical variablesPreanalytical variables contdcontd

Histology LabReceivingProcessingMicrotomyMicroscope slidesDrying slidesStorage of cut slides

IHC analysis variables

IHC AssayIHC Assay

Pre-TreatmentBlocking

Primary Antibody

Detection System

Chromogen

Counter Stain

Pre-treatmentsPre-treatments EpitopeEpitope Unmasking 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.

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

Pre-Treatment Pre-Treatment contdcontd

ER @ pH6 ER @ 9.5pH

Antigen RetrievalAntigen 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 Shia, Richard J. Cotea, and Clive R. Taylora Journal of Histochemistry and Cytochemistry, Vol. 45, 327-344

BlockingBlocking

Endogenous Peroxidase

Endogenous Phosphatase

Avidin, Biotin

Serum Block

Protein block

Primary AntibodyPrimary AntibodySelection of Ab or Panel of Abs

Types of AbPolyclonal Vs MonoclonalRabbit Vs Mouse Monoclonal

– Titration / Time assay: Checker-boardAb and/or SecondaryConc and TimeTemperature: RT, 37*C, 4*C

IHC variablesIHC 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,TdTAgitation: 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

Cervix NordiQC1. Insufficient HIER

2. Clone not robust

3. 10 / 20 Too low conc

4. Epitopes lost

ER Sub-optimal stainingER Sub-optimal staining

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 CloneAb Clone

Ab problems & solutionsAb problems & solutions

Monoclonal vs PolyclonalDiluentAffinity and BondingAb reaction timeStorage

DAKO

Polymer Based 2 Step:ENVISIONPolymer Based 2 Step:ENVISION

DAKO

Labelled Streptavidin-Biotin(LSAB)Labelled Streptavidin-Biotin(LSAB)

Counter StainCounter Stain

HematoxylinLight GreenMethyl Green

Chromogen (Substrate)Chromogen (Substrate)

PeroxidaseAECDABVector S-GVector-VIPVector Nova Red

Alk PhosNew FuchinFast RedBCIP/NBTVector RedVector Black

ControlsControls Poistive and Negative

Tissue Controls / Cell PelletsReagent ControlAb Isotype Controls

Trouble ShootingTrouble Shooting

1. No staining

2. Weak Staining

3. Background

4. Tissue falling off

5. Edge Effects

6. Artifacts

7. And million others…..

Ab DilutionAb Dilution SC SC WC = DF 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 VolumeAV – Ab Volume

What you need in the IHC LabWhat you need in the IHC Lab

Consumables:– Ab– Detection Systems– Chromogen– Buffers– Antigen Retrieval Reagents

Instruments / EquipmentsInstruments / Equipments

IHC Stainer:Antigen Retrieval

– Water Bath– Pressure Cooker– Microwave Oven

Cost of Starting IHC LabCost of Starting IHC Lab

PlaceLabour - TrainingCost of EquipmentsCost of Ab and ReagentsCost of Consumables

http://pri.dako.com/28252_er-pr_pharmdx_interpretation_manual.pdf

http://pri.dako.com/28252_er-pr_pharmdx_interpretation_manual.pdf

H-score The H-score is a method of assessing the extent of nuclear immunoreactivity, The score is obtained by the formula:

3 x percentage of strongly staining nuclei

PLUS 2 x percentage of moderately staining nuclei

PLUS percentage of weakly staining nuclei,

giving a range of 0 to 300.1Ishibashi, H., T. Suzuki, et al. (2003). "Sex steroid hormone receptors in human thymoma." J Clin Endocrinol Metab 88(5): 2309-17.

 

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