ihc technology
DESCRIPTION
It is easy to follow an established protocol recommended by the manufacturer. However that protocol need to be fine tuned for your lab to save money and time. If you have the luxury of time you may be able to save a bundle by staining using a very diluted antibody by staining overnight or even extending incubation time for one hour.TRANSCRIPT
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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