Fixation procedure? Fixation procedure. Formaldehyde fixation. Formaldehyde fixation. Seminoma: Biology or Artefact? ( CLSI ) PMS2, EPR3947

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1 Centrl Prmeters in Optimiztion Strdiztion of Applied Immunohistochemistry The totl test prdigm Immunohistochemistry is techniclly complex, no spect of this complexity cn be ignored, from the moment of collecting the specimen to issunce of the finl report Tylor CR. Arch Pthol Lb Med 2; 124:945 Prenlytic Prenlyticl prmeters Prefixtion Fixtive Fixtion Postfixtion Processing Dehydrtion clering Prffin impregntion Prffin sectioning Storge Ole Nielsen Dept. of Pthology Odense University Hospitl Anlytic Interpretive Epitope retrievl Blocking Primry Antibody Dilutent Detection system Chromogen Counter stin Mounting Design of controls Positive controls Negtive controls Interprettion Criticl Stin Indictor Declcifiction: Type, Time, Temperture Seminom: Biology or Artefct? Seminom: Biology or Artefct? of fixtion? Center Edge PMS2, E3947 MSH6, EP49 PMS2, E3947

2 Bsed on our findings, it ppers tht regrdless of the ntibody clones evluted, fixtion hs negtive effect on hormone receptors. //H2 cold //H2 cold IZ Yildiz-Akts et l in fixtion:.5 in fixtion:.5 IZ Yildiz-Akts et l in fixtion: 3 in fixtion: 3 Appl Immunohistochem Mol Morphol. 211 Oct;19(5):46-9. //H2 cold //H2 cold IZ Yildiz-Akts et l in fixtion:.5 in fixtion:.5 in fixtion: 24 in fixtion: 24 in fixtion: 24 in fixtion: 24 IZ Yildiz-Akts et l Liver, kidney, spleen, colon brest Non-refrigerted smples re ffected more by prolonged cold thn refrigerted smples. period of s short s one-hlf hour my occsionlly impct the immunohistochemicl (IHC) stining in fixtion: 48 in fixtion: for48progesterone receptor. Significnt reduction in IHC stining for hormone receptors, H2, however, in fixtion: 48 in fixtion: 48 generlly does not result until 4 h for refrigerted smples 2 h for nonrefrigerted smples. The ASCO/CAP guideline of cold period of 1 h is prudent guideline to follow. in fixtion: 3 in fixtion: 3 3 Sme cse s 1. cse The s tumor stined s tumor 2 þ for H2s(sme biopsy) h ofbiopsy) but fixtion (), but 3 Sme 1. The stined 2 þ s for H2 (smets.5 t fixtion.5 h of (), demonstrted milddemonstrted reduction in stining t 3 h (b) ws completely negtive t 24 h (c)negtive 48 ht(d). photomicrogrphs were tken mild reduction in stining t 3 h (b) ws completely 24All h (c) 48 h (d). All photomicrogrphs were tken t " 2. t " 2. H-ss 4þ Averge H-ss 33 Averge fors 4 Averge H-ss for 33 Averge Sme cse s 1. cse The tumor stined s 2 þh-ss for H2 (sme biopsy) ts.5 h ofbiopsy) (), but fixtion Sme s 1. for The tumor stined 2 sfor H2 (sme t fixtion.5 for h of (), but cold mild cold forinrefrigerted cold for (t room tem reduction forall non-refrigerted (t room temdemonstrted reduction stining t 3smples h for (b) ws completely negtive t 24 h (c)cold 48 ht (d). photomicrogrphs were tken demonstrted mild inrefrigerted stining t 3smples h (b) ws completely negtive 24All hnon-refrigerted (c) 48 h (d). photomicrogrphs were tken perture) smples perture) smples t " 2. t " 2. H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s H-s 3 H-ss 4 Averge H-ss 3 for H-ss for 4 Averge for H-ss period (h)averge period (h)averge for cold cold for refrigerted smples cold for non-refrigerted room tem for refrigerted smplesperiod cold for(t non-refrigerted room tem(h) period (h) (t perture) smples perture) smples.5 193; ; ; ; 15 H-s H-s.568 H-s.9361 H-s H-s H-s.568 H-s.9361 H-s 1 2; ; ; ; ; ; ; ; H-s H-s H-s H-s H-s H-s H-s H-s 2 194; ; ; ; ; ; ; 122; period (h) 19;3period 22 (h)12; ; 21 15; 178; ; 22 12; ; ; ; 8 146;3period 18 (h) 87; ; ; period ; 18 87; (h) ; ; ;417 78; 5 159; ; ; ; ; ; ; ; ; ; 2 115; ; 2 145; ; ; ; ; ; ; ; ; ; ; ; ; ; ; 9 63; ; ; ; ; ; ; ; ; ;322 12; ;221 15; 178; ; ; ; ; ; 8 146;318 87; 7 182; ; ; ; ; ; ;417 78; 5 159; ; ; ; tumor on single cse of unequivoclly H2tumor on single cse of unequivoclly H ; ; 2 115; ; 2 145; ; ; ; tht,63; positive tumor. Therefore, results re They tht9for,63; fixtion s positive tumor.thetherefore, the not resultsthey not 118; 48 re found for found 118; 2 fixtion s ws less 1D5 less thn 6F11 1D5 clones. pplicble to most clinicltospecimens. on thn SP1 ws 6F11 clones. pplicble most clinicl specimens. effect on SP1 effect effectinto of the The SP1 clonethe hdsp1 more intense stining, Qiu et l27 looked looked effect of clone hd nucler more intense nucler stining, Qiu etinto l27 the fixtion on three clones of clones low bckground, no cytoplsmic stining when stining when fixtion on three of low bckground, no cytoplsmic tumor6f11, on (1D5, single cse of unequivoclly H2tumor on single cse of unequivoclly H2(1D5, SP1)6F11, (PgR636, 16, 1E2) 1D5 6F11 clones. Similrly to Similrly to SP1) (PgR636, 16, 1E2) 1D5 6F11 clones. They found tht for, fixtion s positive tumor. Therefore, the results re not 24 They found tht for, fixtion s positive tumor. Therefore, the results re not 24 on the sme group of sme cses group s thtofofcses Khoury et l.of Khoury, it fixtion hd fixtion on the s tht et ws l. found,tht it ws found tht hd 1D5 less thn 6F11 1D5 clones. pplicble to most clinicltospecimens. effectless on thn SP1 ws 6F11 clones. pplicble most clinicl specimens. effect on SP1 ws effectinto of the The SP1 clonethe hdsp1 more intense stining, Qiu et l27 looked looked effect of clone hd nucler more intense nucler stining, Qiu etinto l27 the Modern Pthology Modern (212) 25,Pthology (212) 25, fixtion on three clones of clones low bckground, no cytoplsmic stining when stining fixtion on three of low bckground, no cytoplsmic when (1D5, 6F11, SP1)6F11, 1E2) 16, 1D5 6F11 1D5 clones. to Similrly to (1D5, (PgR636, SP1) 16, (PgR636, 1E2) Similrly 6F11 clones. on the sme group of sme cses group s thtofofcses Khoury et l.of24khoury, it fixtion hd fixtion hd on the s tht et ws l.24 found,tht it ws found tht Results - Morphology IHC Fixtion dely Modern Pthology Modern (212) 25,Pthology (212) 25, Prenlytic vrible Despite the differences in the rte of loss of epitope integrity in this study, the results from the multiple IHC nlysis consistently supported the overll conclusion tht cooling t 4 C preserves tissue in contrst to vcuum seling which hs no tissuepreserving effect. Recommendtions Recommendtions ASCO/CAP Fixtion dely I Less thn 1 hr Less thn 12 hrs 4 C is better thn RT Engel KB, Moore HM. Arch Pthol Lb Med. 211;135:

3 Seminom: Biology or Artefct? Fixtion procedure? + A long list of experts dvisors Center Edge ( ) PMS2, E3947 Fixtion procedure Fixtive Fixtion Formul Concenttion Postfixtion Tissue to fixtive rtio Method (Immersion, MWO, soniction, movement etc) ph Wshing conditions durtion Storge regent durtion Formldehyde fixtion Phse 1 Penetrtion Very fst Phse 2 Binding Very slow Phse 3 Cross-linking Slow Formldehyde obey the diffusion lws, tht is, the depth penetrted is proportionl to the squre root of. Penetrtion rte cn be determind using the eqution: d = K t Time Tempertur d = Distnce penetrted in mm K = Medwr s coefficient of diffusibility t = Time in Formldehyde fixtion Medwr s K = 5,5 Bker s = 3,6 Biotechnique Histochemistry. 1994; 69, Penetrtion rte cn be determind using the eqution: d = K t Using Bker s K = 3,6: Fixtion: 17.6 mm 5 mm 1 second 1 minute 4 minutes 16 minutes 1 hour Medwr s K = 5,5 Bker s K = 3,6 d =.6 mm (>3 mm/hr) d =.465 mm (27.88 mm/hr) d =.93 mm (13.94 mm/hr) d = 1.86 mm (6.97 mm/hr) d = 3.6 mm (3.6 mm/hr) d = 7.2 mm (verges to 1.8mm/hr), d = 1.18 mm (verges to 1.27mm/hr), d = 14.4 mm (verges to.9mm/hr), d = 17.6 mm (verges to.73mm/hr), d = 35.3 mm (verges to.36mm/hr). 4x4x4 mm liver tissue App. 25 hrs 1% formldehyde binding fter pp. 25 hrs

4 Seminom HE PMS2 Biotechnique Histochemistry. 1994; 69, x4x4 mm liver tissue Dvid G. Hicks App. 25 hrs 1% formldehyde binding fter pp. 25 hrs Edge PMS2, E3947 fixtives Center Am J Clin Pthol 23;12:86-92 Clone E3943 cn not be used on lcohol-fixed tissue Bouin 24 hrs Crnoy 24 hrs Zmboni 24 hrs Clrke 24 hrs Methcrn 24 hrs Form/Zn 24 hrs NBF168 hrs NBF 48 hrs Ethnol 24 hrs NBF 6 hrs Alterntives to 4% NBF... Nme Contins... Compny F-solv Dent. EtOH / Aldehyde derivte / Stbiliser Yvsolb UPM Ethnol / Methnol / 2-Propnol / Formldehyde Copn GreenFix Ethil / Ethnol Dipth CyMol Ethnol / Methnol / 2-Propnol Copn RCL-2 Ethnol / Acetic cid / Complex crbohydrtes Alphelys FineFix Ethnol / Glycerol / PVA / Simple crbohydrtes Milestone Formldehyde-EtOH Formldehyde / Ethnol / Buffer BBC Biochemicl Zn-Formlin Formldehyde / Methnol / Zn-sulfte Richrd-Allen Prefer Glyoxl / Ethnol Antech Dvidson s AFA Formldehyde / Ethnol / Acetic cid Electron Micr. Sci. Moleculr Fixtiv Methnol / Polyethylenglycol Skur Pen-Fix Formldehyde / Ethnol / Buffer Richrd-Allen Histochoice Glyoxl / Zn-sulfte / Butil Ameresco-Inc. 99 % Concordnce for O-Fix Formldehyd / Ethnol / Acetic cid SurgiPth 95 % Concordnce for GTF Glyoxl / Ethnol SttLb Medicl 98 % Concordnce for H2 PAXgene Tissue-fix Alcohols / Acid / A soluble orgnic compound Qigen- PreAnlytix Fixtion i 4% NBF for 13 versus 79 Concordnce between short fixtion long fixtion:

5 4% NBF versus FineFix FF 24 hrs Streck s tissue fixtive (STF) We found tht STF significntly enhnced the stining intensity of phosphoproteins 1% or 4% PFA. Our results indicte tht the choice of fixtive could significntly ffect the usbility of clinicl tissue smples for evluting phosphoprotein by IHC. 25 With existing IHC-protocols 35% (9 of 26) of the ntibodies gve poor or borderline rections on tissues fixed in FineFix Liver - Heprtocyt Ag, clone OCHIE5 (Unpublished dt) 26 Fixtion Prenlytic vrible Recommendtions Recommendtions ASCO/CAP I Fixtive formul 4% NBF 4% NBF Time in fixtive 24 hrs* 24 hrs Tissue to fixtive rtio 1:1 1:1 to 1:2 4% NBF = 4% neutrl buffet formldehyde Engel KB, Moore HM. Arch Pthol Lb Med. 211;135: A: hr B: 2 hrs C: 4 hrs D: 24 hrs E: 2+2 *6-48 hrs *8-72 hrs 4% phosphte buffered formldehyde, ph 7, - 7,4 Declcifiction - Results: 24 hrs 4% NBF fixtion prior to declcifiction. 124 ntibodies on TMA s Intensity /+ + EDTA, 1% ph Formic cid (BFA) Method DeclcTM (HCl) Buffet formic cid (BFA): (4M formic cid +.5M N-formit) Reference/No declcifiction: Control FF 24 hrs Brest -, clone 1D5 (4 C) pakt

6 IHC declcifiction Antistof Clone Ref Declc Formic EDTA Elstse, neutrophil, NP57 NP57 CD15, SN6h SN6h + Bcl-2, 124 -Oncoprotein 124 PG-B6p 1,1E+1 + MUM1p + TG14 CD4, 4B12 4B12 CD43, MT1 MT1 TCAR, BF1 -T-Cell Antigen Receptor CD16, 2H7- Fc Gmm Receptor ßF1 2H7 HI186 Bcl-6, PG-B6p CD4, 11E9 Fctor XIII-, poly Oct-1, 12F11 12F11 Oct-2 (C2), poly MUM1, MUM1p -Multiple Myelom Oncogene 1 / IRF4 Bob 1, TG14 III CD52, HI186 Declcifiction CD15, SN6h No declcifiction Formic cid 16hrs EDTA 96hrs No decl + BFA 16 hrs NBF 48 hrs None Declc 4t Formic cid 16t EDTA 96t Declcifiction CD15, 4G11 No declcifiction Fixtion declcifiction in buffet formic cid (BFA) Bcl2, clone124 NBF 6 hrs Declcifiction Elstse, neutrophilic, NP57 Formic cid 16hrs EDTA 96hrs Declcifiction Most ntigens don t survive declcifiction in strong cid (DeclTM) All tested ntigens survive declcifiction in EDTA show no, or miniml reduction in stining intensity Only very few ntigens don t survive declcifiction in formic cid, but pp. 1% show slight reduction in stining intensity lern!

7 Declcifiction Prenlytic vrible Antibodies:. HercepTest b. Clone 4B5 c. Clone CB11 Recommendtions Recommendtions Declcifiction Tissue should be fixed 24 hrs in NBF prior to Interpret cution - declcifiction. ntigens could be lost! EDTA < Formic cid < Strong cid Drying of sections -, SP1 8 C/16 hrs 6 C/6 Procedure for drying of tissue prior to deprffiniztion: The drying temperture should be 6 C for mximum of one hour, 37 C for mximum of 24, or mbient temperture for 24 or longer. 6 C/6 6 C/16 hrs Drying of sections (Bking) Prenlytic vrible Recommendtions Recommendtions ASCO/CAP Drying of sections I 24 hrs t RT or 1 hr t 24 hrs t RT or 5 C - 6 C overnight t 37 C Engel KB, Moore HM. Arch Pthol Lb Med. 211;135: min t 6 C 16 hrs t 8 C Storge 3 months Dry Storge 3 months Wet Dry 3 C 37 C Slide storge 4 C RT Slide storge 2-weeks Slide storge 2-weeks Slide storge RT This study reveled tht indequte tissue processing, resulting in retention of endogenous 4 C wter in tissue sections, results in ntigen degrdtion. Exposure to high humidity during storge results in significnt protein degrdtion reduced immunorectivity, the effects of storge humidity re temperture 3 C dependent. 37 C Wet

8 Storge of specimen 6! 53! 5! p53, pab ! % 4! f r v e!t 3! -8 C / 3 yers 32! PAb181/cit 25'! 4 C / 3 yers 2! Prenlytic vrible 2! Recommendtions ASCO/CAP 1! I Recommendtions! Estrogen Receptor, 1D5 Ny! New -8 C! 4 C! 2 C! Storge Opbevring!, 1D5 5! 45! 46! 46! Storge of prffin blocks Indefinitely < 25 yers Storge of sections (slide) 7 dys or < 6 weeks < 6 dys 4! % 35! Engel KB, Moore HM. Arch Pthol Lb Med. 211;135: f 3! r v e t! 25! /cit 25'! 2! 15! Storge t -8 C gve superior results 1! 1! 5! 5!! Ny! New -8 C! 4 C! Storge Opbevring! 2 C! Dys...2 C Weeks...4 C Months...-2 C Yers...-8 C Conclusions Less thn hlf of the identified prenlyticl vribles in IHC hve been exmined in published reserch The mjority of tested prenlyticl vribles impct the finl IHC results There is continued need for rigorous reserch comprehensive guidelines on specimen fixtion, processing, storge Thnk you for your ttention!