IHC Technology
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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.
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IHC Technology
- 1. IHC Technology – Problems Solutions
- 3. IHC Technology Problems and Solutions Lecture Workshop Presented By Lawrence Richards M.S.,H.T(ASCP).,QIHC(ASCP)
- 5. Pre-clinical Variability Assay Variability Scoring Variability GLP QC SOP IHC Assay Development
- 12. 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
- 13. ER @ pH6 ER @ 9.5pH
- 19. 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
- 21. Polymer Based 2 Step:ENVISION DAKO
- 33. 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. 1 Ishibashi, H., T. Suzuki, et al. (2003). "Sex steroid hormone receptors in human thymoma ." J Clin Endocrinol Metab 88(5): 2309-17.
- 34. Thanks to DAKO & Distributor MMJ Biosystems Philippines, Inc . Address: Unit 206 CYA Land Bldg. (formerly LTC Corporate Center Bldg.) #282 EDSA Extension Cor. P. Celle Street, Pasay City, Manila Tel. nos.: (632) 851-0192 to 93/489-1008 Telefax: (632) 853-3665 email: [email_address]
Notes de l'éditeur
- Speaker Intro
- In today’s workshop I will go over IHC staining steps and the problems you may face and how we can solve them. It is my hope that the presentation will fulfill the goal of providing the attendees with an in depth background in technical aspects and quality control in IHC and how to avoid or solve the problem you may encounter.
- All IHC labs are different and what works for me may not be best for your lab. You have to optimize and write a SOP with strict GLP in mind and strictly follow it. Lax of QC is the downfall of many labs. I will go over them as I go thru staining steps. If you understand the theory behind the staining then it is easy to trouble shoot problems and solve them.
- Our goal is to develop an assay that consistently produce the same results to meet the same specification, time after time. Strict adherence to GLP and QC is very important for a reliable, consistent results Consistancy of results is totally dependent upon consistency of METHODOLOGY So, write a SOP and follow it to the word. VARIABILITY 1.Preclinical – starts at Operating Room 2.Tissue Processing – Fixation to slide 3.Assay variability including – Antigen unmasking 4.IHC – Reagents – Ab, secondary, Buffer, incubation time, Temperature 5. Detection method 6. Data analysis – Scoring 7. Interpretation Let us see the Preclinical variables How do we achieve this with so many variables that we do not have any control over? And the ones that we DO have control over.
- We may not have any control over this, but you still have to know. Surgical: that is where quality IHC starts. Cumulative effect on tissue, antigen/ epitope/receptors destruction. How is the specimen removed: laser knife/electric wire loop,needle,forcep piched Bx,cone Bx—all causes damage. Frozen samples. time lapse between surgical removal and into a freezing temperature is critical. Autolysis starts soon after the specimen is removed in surgery. Phospho Ab are becoming very popular since they are very specific. But phosphorylated proteins can rapidly undergo DEPHOSPHORYLATION and become difficult to detect Storage: in OCT compound wrapped in foil @-70*C is ideal Cell Preparation: Air dried or fixed in Acetone, 70% alc,Alc:acetone mix, air dried and stored at –20*C or –70*C FFPE, sooner into fixative less autolysis and arrest dephosphorylation. Radiation and chemo samples and specimen marked with a dye and also exposed for long to OR bright lights or UV lights will have damaged epitopes and less intensity staining.and so should be recorded. Large tissue specimen should be sliced into for fixative penetration before grossing.
- Time lapse in transport if not fixed in surgery. Grossing: sooner the better; fixative ratio 1:20; size <2mm slabs More rigid cell structures like SqEpithelium and fibrotic capsules should be removed or sliced into for fixative to fix interior. Fatty tissues like( Breast needs 8hrs to 48hrs )need longer fixation time, bloody tissues need change of fixative often. FIXATIVE: 10%NBF is the best.Not more than 48 hours, longer into 70%alc. Min 6hrs to 48hrs. Chemistry takes 6hrs not the size of Bx B5/Zenkers mercurial-bad for CD5,CD30,CD23, Bouins picric CD5,CD10,Cyclin D1, Hollande-by Gis-most lymphoi related epitopes destroyed. Zinc formalin good for CD5,CD10, Some claim Alc fixative can cause leaching of protein and results in diffused staining. Eg: FactorVIII Decalcification: reduces antigenicity and results in weak staining. In some cases causes false nuclear staining especially with avidin biotin systems. STORAGE: away from heat, in 70%alc for long storage after 10%NBF fixation.
- When recd note: Fixative to specimen ratio 1:20,any drying observed,autolysis observed,bloody and very fatty specimen needs washing and trimming off fat before processing. Max thickness 2mm slabs for good processing. Process with heat only in paraffin and wax temp @ 60*C and total 2 hour in wax. Use paraffin with polymer for cutting thin sections.Do not have Bx samples with surgical large samples,Bx needs less time in processor. Microtomy: get section w/o compression, use good sharp knife. Steel knife vs disposable knife. Do not use any adhesive in the water bath. Microscope slides: use poly-l-lysine coated slides or positively charged. Drying slides: do not use heat more than 60*C. Most Ab are heat sensitive(dry) not when in buffer HIER. Eg: ER/PR , GCDFD-15 heat labile. The best is o/n at 36*C and 1 hr in 60*C just before staining. BE CONSISTANT. Storage: store cut slides in 4*C, for long time coat them with paraffin and then 4*C
- Problems are there in every step of the way. We will see how we can detect, avoid and solve them as we go along.
- #1 call to Tech service is about antigen Retrieval Antigen Retrieval / Epitope Unmasking / Target Retrieval / Antigen Enhancer Target retrieval system United States Patent 4890847 Two types use of Proteolyitic enzyme digestion and Heat Induces Epitope Retrieval (HIER) Enzymatic Digestion : epitope unmasking: The first one used to enhance staining. Trypsin: (Light-Med)at 37*C for AE1/AE3,B72.3, Factor XIIIa, HAM-56 I use 0.025% for 1 min after HIER for bringing up membrane staining 0.1% for 3 min is normally used, but again it differs from lab to lab. Pepsin(Strong) @ 37*C for CK7,CK20,Pan CK,Lu-5(7’) Protease 24(Extra Strong)@RT for EGFR(5’) Protinase K (Very very strong) watch it it will eat away your tissue.(5’) Conc x time study necessary Caution: While Trypsin is good for Skin, Pepsin is not. Use correct enzyme or you will destroy it. All the glandular cells will look empty( horses have left the coral) David Tacha, Biocare HIER : Retrieval soln: NaCit pH 6;Tris Buff pH9.5;EDTA pH8, measure pH Increased Endogenous BIOTIN artifact by ABC method. Biotin blocking, background. Temp: 95*C (water bath40/20) Steamer <100*C30-60’/10’and Pressure cooker 120*C 3’/10’@15lbs pressure NOT good for HMB45,MOC-31,mesothelin. Microwave 100-105*C can also be used- caution drying due to evaporation.20’ in buffer/20 cool Caliberation necessary, hot spots, tissue loss due to boiling Always keep same number of dishes and racks with fluids. KEEP IT the SAME everytime: Consistancy
- Each Ab needs different TRS with different pH AND different Temperature and Time for best performance. This is the most important step and I am going to spend more time on this. Ki67
- We need to block endogenous peroxidase activity for HRP system with H2O2 and Phosphatase activity in Alk Phos systems with levamisole. In ABC method endogenous Avidin and Biotin should be blocked. Dry milk and egg white are used by some. It is cheap to buy RTU.Biotin artifact after HIER is very troublesome, it can be extremely intense mimiking true staining, even localized only in tumor cells with crystal clear stroma. Serum: I found better and cleaner results when this step is omitted. Some add serum to the Ab diluent. If we miss H2O2 where will u find staining RBC,Granulocytes,Eosinophils,hepatocytes,Muscle,Kidney,Monocytes. Alk Phos is destroyed during paraffin processing.So does not need for FFPE but needed for Frozen. Interesting observation: you can do H2O2 after secondary also. Protein Block:Reagents used to reduce the chances of nonspecific reactions of an antibody with components other than its target antigen. There are many RTU products available in the market if you want to use them. We use them only for frozen.
- There is no perfect marker for any tumor: that is to say there is no such thing as an antibody with 100% sensitivity and 100% specificity for a given cell or tumor. NSE is not enough to dx neuroendocrine ca.EMA+lymphoma+? Misleading Selection of clone(monoclonal) or polyclonal is upto your requirement. Some like some clone and the other another clone.If the sensitivity of monoclonal is low then we go for poly. With Rb monoclonal in the market there are lot of choices. Poly tend to have more affinity binding so seldom get washed off. Sometimes monoclonal cocktails are used with great success. Eg AE1/AE3. Ab should be selected to be compatible with your detection system. It is too expensive to have more than two detection systems in the lab. We keep Dual Ms/Rb Envision and a Goat secondary system for goat Ab. 1:100 to 1:2000 Poly antisera,1:10 to 1:1000 for Mouse cell culture supernatant,1:000,000 for monoclonal Ab from Ascites fluid Dilution x time checker board method is the best for optimizing dilution and incubation time. Many labs stick to 30’ or 60’ and titrate their Ab. They also keep the (secondary) detection system constant. Incubation: 37*C enhances staining intensity and therefore lower titer can be used.But background also increases. Samething with overnight at 4*C, 10 to 100 fold increase is possible. Watch out for drying. Keep the tray level! Problems with Ab and how to avoid and solve is my next slide
- Here are some variability of a few Ab to different treatments. 37*C reduces staining of ALK1,CD3,CD4,CD10,CD5,CD43,CD459UCHL-1),Cyclin-1,ER,ER1D5(does not work or weak),MIB1,PR,TTF-1,TdT ER(1D5) over agitation dislodges antibody binding from Epitope Use of Ab diluent (Background reducing Diluent=Serum, Ab stains weak. But less background.
- 1D5 stronger than 6F11 or SP1, there are new clones released frequently like H222, LH2 etc It is like buying a new car, you do not want to buy until the review comes out.. You do not know the characteristic of these new clones until we have adequate published information availabel. Epitope lost in excess washing, weakened by high heat, decalcification, wrong fixative, too long in fixative.
- LMW CK cocktails with different staining patterens.
- How it is prepared matters-purification with hi salt or hi temp will affect Ab performance. Monoclonal – specific to one epitope and less non specific binding, selection of clone is important.Since one kind of epitope, fixation is critical.Depend more on environmental factor such as pH and salute for optimum performancePoly; more accomodative with harsh treatment. Diluent: Most important. Follow Mfr., pH 7.4 and correct buffer is critical. PBS with 0.2%BSA is a diluent used by us brefore we switched to DAKO background reducing Ab dikuent. Time required to reach equilibrium with the antigen. Ionic interaction, H bonding and Van der Walls force only = no covalent bonding. THE REACTION IS REVERSIBLE. Dissociation during washing. Poly withstands better. Factors that weaken bond are hi salt conc, hi temp, v.low pH. Reactivity: depends on fixation,titre,temp,movement,detection system. Titre: too strong less or no staining = competition for antigen sies and weak binding when more than one Ab binds with one Ag. Storage: follow mfr, no freeze-thaw, aliquot. IgG2a v.sensitive.-20*C.return Ab to cold.wear gloves-do not allow microbes to get in. Remember Mfr are required to test shelflife for a fixed time only, they may be good for a year or more from exp dt. BUT test every month.
- An unlabelled Primary is first employed, the second reagent is a dextran polymer to which are bound large number of peroxidase molecules and secondary molecules to bind to the bound, unlabelled primary. This system and similar ones are better in the sense that there are no biotin artifacts.
- Biotinylated Secondary Antibody links the Primary and the Streptavidin peroxidase conjugate.
- Hx is used in most of the time. Harris with TBST bluing is what we use and it gives excellent nice blue. Avoid reddish color Hx that can cause diagnostic problems. Also that tend to give a violet background color. Light green and Methyl green are very good for nuclear stains,(ER) but tend to streak. Keep Hx stain very light you have a universal counter stain.
- HRP system use DAB, AEC and Vecta Red , AEC no xylene……, In US it is important to save stained slides for 10 years. So they need to be permanently coverslipped. As of now DAB and permanent RED are the only chromogen used in most of the labs. Alk Phos uses Fuchsin, Fast Red and NBT. This system is not very popular in US. The staining fade over the years. However in research institutions for multi Ab staining, this method is necessary.
- Control for doing AR and titration: Multitumor block with known sensitivity of 1+,2+ and 3+ is essential for optimizing Ab and AR method selection. The time spent in this effort is enormous. If you make a few blocks you can use them as +control block everytime you do IHC run. A 2mm thick tissue block can yield 300 to 400 slides. Store the slides in 4*C and for long term storage dip in molten wax and store them in 4*C. A multi block should have normal tissue and tumor tissue you normally come across in your lab. Should have Isotype of Ab as Neg control, to see tru pos. If u have 4 different Isotype Ab u should have 4 controls. Do not think running a control is waste of reagents. It will save a lot of wrong diagnosis and headache of guessing what went wrong. You need a control block to test every new lot of Ab along with the previous lot before use.
- This is a general trouble shooting; All mfr provide you a similar chart No staining: A step omitted,excess dilution,incubation time too little,reagents expired,Ab-An complex washed away,incorrect AR,chromogen not solvent resistant,chromogen not made correctly. Weak : expired reagents,incorrect buffer,antigen level too low,too hi con of Ab,used PBS in Alk Phos. fixative permanent damage,preclinical reasons,dewax not complete,AR temp not correct,used dull knief causing serum protein to diffuse.necrotic tissue,improper fixation. Background: “Blush”,”Diffused” endogenous biotin enhanced by HIER,too high conc,chromogen not made correctly,improper rinsing,too long wash in tap water,wrong serum blocking,dewax not complete,very thick section,microbial contamination,bloody specimen,crushed specimen,necrotic,slide adhesive not the right one for IHC,antigen diffusion prior to fixation, antigen smearing due to dull knife.cross contamonation with positive Ab in a neg slide,drying effect,non specific staining in fatty tissue.Hydrophobic and ionic interaction.excess fixation. Non buffered formalin used. False Positive:Beware of the correct signal in the correct place.But can result from ‘cells producing,binding,phagocytosing,actively transporting the specific An. Tru but undesirable- PR sticking to mucin, or hemosiderin/pigments/virus-bacteria. Stining on sticky cells eg.,islet cells,Leydig cells.
- 1:100 OR 10ug/ml (stock soln 1000ug/ml) When manufacturer makes new @ 1200ug/ml? 1:100 = 1:120? 1ul+99ul=1:100(stock 1000ug/ml) 1.2ul+98.8=1:100(stock 1200ug/ml) 1ul+99ul=10ug/ml(1000ug/ml) 0.83ul+99.17ul=10ug/ml(1200ug/ml)