Immunohistochemistry (IHC) Optimizing IHC protocols. Cyclin D1 in MCL. What can possibly go wrong?! IHC protocol. Optimizing IHC protocols

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1 Immunohistochemistry (IHC) Optimizing IHC protocols M Ole Nielsen Dept. of Pathology Odense University Hospital Nov Tissue/Cells M Lab 1 Cyclin D1 in MCL Lab 2 Tissue/Cells Lab 3 Lab 4 What can possibly go wrong?! Optimizing IHC protocols IHC protocol Blocking of endogenous activity Epitope retrieval Primary antibody Detectionssystem Chromogene/substrate Counterstain Mounting of cover slips

2 Solut y - Infinite logy Toda ote D ess, Rem s Techno Random Acc Continuous Tomorrow' ope, e-to-microsc otom t-once, Micr One, All-A Xmatr x Immunostainers r ne tai S to Au Immunohistochemistry (IHC) ULTRA Immunohistochemistry is technically complex, and no aspect of this complexity can be ignored, from the moment of collecting the specimen to issuance of the final report BenchMark Ramon CA Road,San ris Canyon : Nor , Fax Taylor CR. Arch Pathol Lab Med 2000; 124:945 Bond Sample Tissue Cells Smears, Cytospin... Fixation LBC Dehydration Drying Imprint Fixation Paraffin embedding Storage Fixation Buffer Cutting Fixation Buffer Mounting Buffer Freezing Fixation Cutting (Decalcification) Drying Optimal protocol Sensitive detection system Good primary antibody The right conc. (dilution) of primary Choose the best retrieval method Drying Deparafination Test material: Hydration IHC protocol Control slides Detection systems 1 PAP Test Control Multiblok 2 APAAP PowerVision+ LSAB Super Sensitive ABC NovoLink/Bond Polymer 1 Dako 2 ImmunoVision Tech. (AH) 3 BioCare (Prohosp) 4 Zymed (AH) 5 BioGenex (Prohosp) 6 Lab Vision (AH) 7 Vision Biosystems (TriChem/Leica) 8 Ventana, Roche 9 CellMarque Advance/EnVision Mach2/Mach3/Mach4 4 Picture UltraVision 6 UltraView/OptiView TSA/CSA HiDef 9 8

3 LSAB and ABC LSAB vs EnVision+ Biotin HRP StreptAvidin CD34/LSAB/AEC CD34/EnVision+/DAB+ Endogenous biotin Polymer and multimer systems Ventana BenchMark Leica Bond Dako AutoStainer PowerVision+ vs EnVision Flex+ PowerVision+ EnVision Flex+ OptiView UltraView PowerVision SuperSensitive Mach4 Bond Polymer EnVision (Dextran) Picture (Polypeptide) UltraVision (Polypeptide) OptiView vs EnVision Flex+ Tyramide signal amplification (TSA/CSA) OptiView HQ: Hydroxy-quinoxaline EnVision Flex+ What is Tyramide? Phenolic compound Tyramine NH2 In presence of HRP, catalyzes the generation of a free radical Binds to electron rich moieties of proteins (i.e. tyrosine); Only within diffusion length of the free radical. HO Can be used as potential method of amplification when covalently bound to a hapten

4 OptiView + Tyramide ampl. OptiView + Tyramide ampl. OptiView OptiView HQ: Hydroxy-quinoxaline HQ: Hydroxy-quinoxaline HQ - Tyramide HQ - Tyramide H2O2 H2O2 OptiView + Tyramide ampl. CSA II vs PowerVision CD133 in Seminoma OptiView HQ: Hydroxy-quinoxaline HQ - Tyramide CSA II PowerVision+ UltraView Amp Vs OptiView OUH Afd. f. Klinisk Patologi O. Nielsen OptiView Vs OptiView Amp 1:10_Prot2-4-CC _UVamp 1:10_Prot2-4-CC _OV Muscle SARCd, clone 12C1 Muscle SARCd, clone 12C1 OUH Afd. f. Klinisk Patologi O. Nielsen 1:10_Prot2-4-CC _OV 1:10_Prot2-4-CC _OVamp4-8

5 OUH OptiView Vs OptiView Amp Afd. f. Klinisk Patologi O. Nielsen Pancreatic adenocarcinoma Fixation and processing SMAD4_EP618Y_50_32 CC _OV SMAD4_EP618Y_50_32 CC _OVamp Fixatives NBF fiksering Aldehydes Formaldehyd Glutaraldehyd Glyoxal Coagulating fixatives Ethanol Methanol Acetone Picrinsyre Mercuriklorid Chromtrioxid Fixation Fixation and IHC Aldehyde Coagulating Coagulative fixation Aldehyde fixation Crosslinking

6 Fixation of cells. TdT, poly on MOLT4 CD20 and fixatives - Acetone Ethanol Metanol Bouin 24 hrs Zamboni 24 hrs Clarke 24 hrs Ethanol 24 hrs A/M NBF Carnoy 24 hrs Methacarn 24 hrs NBF-Triton Form/Zink 24 hrs NBF 6 hrs NBF168 hrs NBF 48 hrs NBF 24 hrs CD79a and fixatives CD79a and epitope retrieval Bouin 24 hrs Zamboni 24 hrs Clarke 24 hrs Bouin 24 hrs Carnoy 24 hrs Methacarn 24 hrs Form/Zink 24 hrs Ethanol 24 hrs NBF168 hrs NBF 48 hrs NBF 24 hrs Zamboni 24 hrs Clarke 24 hrs Carnoy 24 hrs Methacarn 24 hrs Form/Zink 24 hrs Ethanol 24 hrs NBF 6 hrs NBF168 hrs NBF 48 hrs NBF 24 hrs NBF 6 hrs Hepatocyt Antigen, OCH1E5 Anbefalede fikseringstider* * DSPAC NBF 3t + For at opnå bedst mulig kvalitet af de immunhistokemiske farvninger, skal vævet fikseres i 24 til 48 timer. Operationspræparater defineres som præparater, hvor der normalt NBF 48t

7 Fixation i 4% NBF for 13 hours versus 79 hours Concordance between short fixation and long fixation: 99 % Concordance for ER 95 % Concordance for PR 98 % Concordance for HER2 Alternativer til 4% NBF Navn Indhold Firma ISH * F-solv 2 Denat. sprit / Aldehydderivat / Stabilisator Yvsolab - UPM 3 Ethanol / Methanol / 2-Propanol / Formaldehyd Copan + (24t) GreenFix 3 Ethandial / Ethanol Diapath + (24t) CyMol 3 Ethanol / Methanol / 2-Propanol Copan + (24t) RCL-2 2 Ethanol / Eddikesyre / Komplekse Kulhydrater Alphelys + (24t) FineFix 2 Ethanol / Glycerol / PVA / Simple Kulhydrater Milestone + (24t) Formalin-Sprit 1 Formaldehyd / Ethanol / Buffer BBC Biochemical + (6-48t) Zn-Formalin 1,5 Formaldehyd / Methanol / Zn-sulfat Richard-Allen + (6-48t) Prefer 1,4 Glyoxal / Ethanol Anatech + (24-48t) Davidson s AFA 1 Formaldehyd / Ethanol / Eddikesyre Electron Micr. Sci. - Molecular Fixativ 5,7 Methanol / Polyethylenglycol Sakura + Pen-Fix 4,5 Formaldehyd / Ethanol / Buffer Richard-Allen +/- Histochoice 4 Glyoxal / Zn-sulfat / butandial Ameresco-Inc. - O-Fix 4 Formaldehyd / Ethanol / Eddikesyre SurgiPath + GTF 4 Glyoxal / Ethanol StatLab Medical - PAXgene Tissue-fix Alcohols / Acid / A soluble organic compound Qiagen- PreAnalytix + 1 Reference * Fikseringstid OUH Afd. f. Klinisk Patologi O. Nielsen FineFix Afdeling for Klinisk Patologi, OUH Materiale Afdeling for Klinisk Patologi, OUH Væv (14) Fiksering Antistoffer (26) Ethanol Propylenglycol Glycerol Polyvinyl alkohol Monosaccharider 1 Ethanol 99% 720ml 2 Stamopl. 280ml Tonsil (4t - 24t - 48t) Rectum Lever Lunge Mamma (x2) Binyre Renalcellecarcinom Rectum adenocarcinom Hepatocellulært carcinom Lunge adenocarcinom Thymom NBF 4% : 4t - 5dg Ethanol 77% : 4t - 5dg FineFix : 4t - 5dg CD45, 2B11+PD7/26 DESMIN, D33 CEA, Col-1 CK, AE1/AE3 TTF-1, SPT24 CK7, OV-TL12/30 CK 18, DC10 CK 20, Ks20.8 CD20cy, L26 CD10, 56C6 CD19, LE-CD19 CD3, F CD5, 4C7 CDX2, ATM28 MLH1, G PgR, PgR636 CK, CAM 5.2 CK14, LL002 GCDFP, D6 MSH2, 27 INHIBIN, RI MGB1, 304-1A5 ER, 6F11 HEPATOCYTE, OCH1E5 KAPPA-SmIg LAMBDA-SmIg

8 Afdeling for Klinisk Patologi, OUH Afdeling for Klinisk Patologi, OUH Lungecarc. CK, AE1/AE3 MBO/TEG Resultater FineFix << NBF Antistof HIER NBF FineFIX HEPATOCYTE, OCH1E5 TEG CK14, LL002 TEG GCDFP, D6 PROT MSH2, 27 EDTA INHIBIN, RI TEG MGB1, 304-1A5 TEG ER, 6F11 TEG n.o NBF 24t FineFix 24t 77% Eth. 24t KAPPA-SmIg Citrat +++ overf. LAMBDA-SmIg Citrat +++ overf. Afdeling for Klinisk Patologi, OUH Afdeling for Klinisk Patologi, OUH Mamma ER, 6F11 TEG60 C/n.o. Lever Hepatocyt Ag, OCH1E5 MBO/TEG NBF 24t FineFix 24t NBF 24t FineFix 24t

9 Antigen/Epitope masking Antigen/Epitope retrieval Coagulative fixation Aldehyde fixation Crosslinking Antigen/Epitope retrieval Antigen demasking (Antigen retrieval / Epitope retrieval ) HIER (Heat induced epitope retrieval) Heating in various buffer solutions Retrieval Non-HIER Pretreatment with proteolytic enzymes Protease Trypsin Pepsin Formic acid (amyloid)

10 Fixeringstid og proteolyse Fixeringstid og proteolyse CK, CAM ,05% Protease Type 14 0,05% Protease Type 14 CK, CAM5.2 0 Planocellulært karcinom (lunge): NBF 6t Protease X min Planocellulært karcinom (lunge): NBF 168t Protease X min. Fixeringstid og proteolyse /91/$3.30 The Journal of Histochemistry 15 CK, CAM ,05% Protease Type Copyright 1991 by The and vol. Cytochemistry Histochemical Society. 39. No. 6. Inc. pp , Printed Rapid Communication Antigen Retrieval in Formalin-fixed, Paraffin-embedded Tissues: An Enhancement Method for Immunohistochemical Staining Based on Microwave Oven Heating of Tissue Sections SHAN-RONG BioGenex Received SHI, Laboratories, for publication MARC San Ramon, January 15, E. KEY, California 1991; and KRISHAN L. KALRA accepted March 12, 1991 (1C2212). Adenokarcinom (lunge): NBF 6t Protease X min. HIER: How does it work? HIER-variables Formaldehyde induced cross links are broken Temperature (60 C C) Retrieve electrostatic charges Time (5 min - 20 hrs) Tissue bound Ca++ are removed Rehydration of the tissue (improved penetration) ph in buffer (ph1 - ph10) Ca++ complexing buffer (EGTA/EDTA) 1991 in USA.

11 B (Ki67) IHC intensity A B C D E HIER and ph ph C (CD43) Epitope retrieval Test battery 1 Non-HIER:! 1. No pretreatment 2. Pronase E (0,05%/37 C)!! Pepsin (0,4%/37 C)!!! 60 HIER-1:! 4. MW/Citrate ph6!!! MW/TE (TEG) ph9!!! MW/TRS (Dako) ph6,1!! 15 TE (TEG): 10mM Tris + 1mM EDTA (EGTA), ph9 TRS: Target Retrieval Solution (Dako S1700) Epitope retrieval Test battery 2 HIER-2:! 7. MW/Ci ph2!!!! MW/Tris-HCl ph10!!! MW/EDTA ph8!!! 15 SYP, Snp88 Why not just follow the recommandations? HIER/PROTEOLYSIS! 10. MW/Citrate ph6!!! 15! PronaseE (0,05%/37 C)!! 2! 11. PronaseE (0,002%/r.t.)!! 8!! MW/TE ph9!!! 15 No AR ( Recommandation ) MW/TEG Is AR always necessary? Protease Neutrophil Elastase MBO/C - AR MBO/TEG MBO/C Insulin, 2D11-H5

12 Laminin, poly Epithel Antigen, BER-EP4 Pepsin Protease MBO/TEG MBO/TEG MBO/TRS Epitope retrieval Test battery 2 HIER-2:! HIER/PROTEOLYSIS! 10. MW/Citrate ph6!!! 15! PronaseE (0,05%/37 C)!! 2! 11. PronaseE (0,002%/r.t.)!! 8!! 7. MW/Ci ph2!!!! MW/Tris-HCl ph10!!! MW/EDTA ph8!!! 15 MW/TE ph9!!! 15 MBO/C ph2 Collagen 4, MAB3 MBO/C ph4 Collagen 4, MAB5 HIER in MW oven Protease MBO/C - Protease

13 Superheating Onboard heating Epitope retrieval Test battery 1 Epitope retrieval Test battery 2 BenchMark Ultra Non-HIER:! 1. No pretreatment 2. Protease 1 (36 C)! 8 3. Pepsin (0,4%/37 C) Offline!! 20 HIER:! 4. CC2, ph6 (91 C)!! CC1, ph8,5 (100 C)!! CC1, ph8,5 (100 C)!! 64 BenchMark Ultra HIER/PROTEOLYSIS! 7. CC1, ph8,5 (95 C)!! 32! Protease 2 eller 3 (36 C) 4! 8. Protease 2 eller 3 (36 C) 4 CC1, ph8,5 (95 C)!! 32!! HIER-2:! 9. MW/Ci ph2 Offline!! 10 CC2: Citrate based buffer, ph6 CC1: Tris-EDTA based buffer ph8,5 10. MW/TRS ph6,1 Offline 15 Low-temperature HIER Low-temperature HIER CK14 MBO/TEG 15 TEG 70 C/16 hrs TEG 60 C/16 hrs MBO/TEG 15 TEG 60 C/16 hrs

14 MBO/TEG CD5 antibodies Fact or artifact? MBO/TRS Clon 4C7 Clon Leu1 Diagnose? SYP, clone snp88 CDX2 CK14 CK20+ CK7- CK5/6+ CK13+ PLAP SYP Pancreas Small intestine /94/$3.30 The Journal of Histochemistry and Cytochemistry Copyright by The Histochemical Swiety, Inc Vol. 42, No. 2, pp , 1994 Printed in US.A. Original Article I Some Ascites Monoclonal Antibody Preparations Contain Contaminants That Bind to Selected Golgi Zones or Mast Cells' SAMUEL S. SPICER,2 M. A. SPIVEY, MAKCMY) I T O, and BRADLEY A. SCHULTE Department of Patbology and Laboratory Medicine, Medical University of South Carolina, CbarZeston, South Carolina. Received for publication April 20, 1993 and in revised form September 17, 1993; accepted September 21, 1993 (3A3001). Ascites antibodies tested Antibody Clone Company Format MAG CK5/6 D5/16 B4 Dako Ascites purified ++ Topo IIa Ki-S1 Dako Ascites purified 0 CK14 LL002 BioGenex Ascites ++++ CDX2 CDX2-88 BioGenex Ascites +++ CD29 JB1a BioGenex Ascites +++ PLAP PL8-F6 BioGenex Ascites ++++ SYP* snp88 BioGenex Ascites ++++ CK13 KS-1A3 Sigma Ascites + CD56 56C05 NeoMarker Ascites purified 0 CD56 56C04 NeoMarker Ascites purified 0 p63 4A4+Y4A3 NeoMarker Ascites purified 0 MAGE-1 MA454 NeoMarker Ascites purified 0 Mouse serum * Clone snp88 was kindly provided by Dept of Pathology, Holstebro Hospital A small proportion of mouse ascites fluid induced by hybridomas producing monoclonal antibodies or myelomas secreting immunoglobulin yielded staining that was confined to the Golgi zone of certain epithelial cell types in rats and gerbils but not in mice. In addition, a commercial IgG fraction from mouse plasma similarly labeled the Golgi area, unlike IgG from mouse serum from another source. Culture supernatant from one hybridoma line contrasted with ascites fluid produced by the same hybridoma in failing to stain the Golgi region. The capacity of a fluid to react with the Golgi cisternae bore no relationship to the class of immunoglobulin secreted by the hybridoma or myeloma. Ab- sorption of an ascites fluid with blood group Ai human erythrocytes eliminated its affinity for Golgi cisternae. Ad- Golgi zone labeling by this ascites fluid. The positive cells included most serous secretory cells in rats, serous cells of sublingual and tracheal glands, and some endometrial and oviduct-lining cells in gerbils, and columnar lining cells of small intestine and cecum and all or part of the lining cells in some prostate lobes in both genera. Some of the tested ascites fluids stained mast cells. The agent accounting for mast cell labeling Wered, however, from that reacting with Golgi cisternae in its distribution among the mouse ascites fluids examined, lack of relationship to the AB0 blood group system, occurrence additionally in normal rat serum, and capacity to stain cells in mice as well as rats and gerbils. (J Histochem Cytochem 42: , 1994) KEY WORDS: Extraneous immunostaining; Blood group; Golgi zone;

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