Antigen Retrieval - NordiQC
Antigen Retrieval - NordiQC
Antigen Retrieval - NordiQC
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<strong>Antigen</strong> <strong>Retrieval</strong>Erfaringer med PT-modul (Dako)Michael Bzorek og Dorthe Thers Strue - NielsenPatologi afd. i Næstved
ProblemPT, High-buffer (3-1 buffer)RT/ 95ºC/85ºC ~41/15/8 min65ºC/95ºC/65ºC ~18/30/32 min65ºC/95 or 97ºC/65ºC ~ 18/20/32 min65ºC/95 or 97or 99ºC/65ºC ~ 18/10/32 minMorfologi ”suboptimal”:• Knoglemarvsaspirater / Koagler• Vabrasio uterus• Celle el. vævsmateriale (Akut / kronisk inflammation)•?
1423<strong>NordiQC</strong>Søren Nielsen /Aalborg
Knoglemarvs aspirate/ Koagel fix. 96h/NBFCD138 clone B-A38PT, High (3-1) 95ºC / 30 min PT, High (3-1) 95ºC / 20 minP/E 65ºCP/E 85ºC
Hepar/Tonsil fix. 24h/NBFCD34 clone QBEND-10HeparTonsilPT,High 95ºC / 30 minS/S 65ºCPT,High 95ºC / 10 minS/S 85ºC
Knoglemarvs aspirat / Koagel fix. 96h/NBFCD5 clone 4C7PT, High (3-1) 97ºC / 20 min v. P/E 65ºCRecommended settings (Dako)PT, High (3-1) 97ºC / 20 min v. P/E 85ºC
Knoglemarvs aspirater (koagler) 8 out of 10 → Dårlig morfologi (65ºC)Granulocyter ?PT, High (3-1) 97ºC / 20 min v. P/ 85ºCPT, High (3-1) 97ºC / 20 min v. P/ 65ºC
PT,High buffer (3-1) v. 95ºC/15 minPre-heat versus End temperaturEnd temp. 85ºCEnd temp. 65ºC† †Pre-heat temp. 65ºC☺☺Pre-heat temp. 85ºC
Effektivitet af forskellige demaskerings procedureTonsil fix. in NBF/24hCD5 clone 4C7PT,T-EG pH 9 95ºC, 30 min - S/S 65ºCPT,High pH buffer(3-1) 95ºC, 30 min - S/S 65ºCMWO, T-EG pH 9, 20 min
HIER procedurerMWO ~ RT / MWO / 75ºC ~ 8 min / 20 min / 10 minPT-Link ~ 85ºC / 95ºC / 85ºC ~ 8 min / 15 min / 8 minPT-Link ~ 85ºC / 95ºC / 85ºC ~ 8 min / 30 min / 8 minPT-Link ~ 85ºC / 97ºC / 85ºC ~ 8 min / 20 min / 10 minSlut titrering:CD5 clone 4C7 1:50 → 1:6400CD23 clone 1B12 1:50 → 1:6400CyclinD1 clone SP4 1:50 → 1:6400PT-Link ~ 65ºC / 97ºC / 65ºC ~ 18 min / 20 min / 32 min (DAKO)
MWO - CD5 1+50 PT-95/15 min. - CD5 1+50 1+6400PT- 95/30 min.CD5 clone 4C7PT- 97/20 min.4,543,5Score32,521,5PT-95/30min.PT-97/20min.PT-95/15min.MWO/TEG/20min.PT- 95/15 min.10,501+800 1+1600 1+3200 1+6400DilutionMWO/20 min.
MWO/TEG/20 min.PT- 95/15 min.Cyclin D1 / SP443,53ScorePT- 97/20 min.2,521,510,5PT- 95/30 min.PT-95/30min.PT-97/20min.PT-95/15min.01+501+2001+8001+1600MWO/T-EG/20min.Dilution
Epitope <strong>Retrieval</strong>Natten over ved 75ºC el. 70ºC (16h)
Epitope <strong>Retrieval</strong>, PT-Link, High ph buffer (3-1) at 75ºC / 16hKnoglemars aspirate / KoagelCD5 CD138 CD3485ºC65ºCPre-heat settings (65ºC versus 85ºC) indflydelse på morfologi / immunreaktivitet
Epitope <strong>Retrieval</strong>, PT-Link, High ph buffer (3-1) at 70ºC / 16hKnoglemarvs aspirat / KoagelCD3485ºC65ºCPre-heat settings (65ºC versus 85ºC) indflydelse på morfologi / immunreaktivitet(billeder fra samme område)
Epitope <strong>Retrieval</strong>, PT-Link, High pH buffer (3-1) at 75ºC / 16hAppendix / Akut inflammationCD13885ºC65ºCPre-heat settings (65ºC versus 85ºC) indflydelse på morfologi / immunreaktivitet(billeder fra samme område)
Konklusion• Pre-heat settings 85ºC (PT-modul) :– Forbedre morfologien ~ Specielt på vise typer af vævsmateriale– Effektiv antigen retrieval ~ Umiddelbart ingen forskel v.65 og 85ºC– Reducerer AR / HIER-tiden ~ 10 - 30min. afh. sluttemperatur– Temperatur eller tidsfaktor ~ Paraffins smeltepunkt (55 - 85ºC) ?
TakMWO : Preheat settings 100ºC ~ 15 min ~ 5 minHigh ph buffer (3-1)Whirlpool / 6.Sense
Epitope <strong>Retrieval</strong>, PT-Link, High ph buffer (3-1) at 75ºC / 16hKnoglemars aspirate / KoagelCD5 CD138 CD3485ºC65ºCPre-heat settings (65ºC versus 85ºC) indflydelse på morfologi / immunreaktivitet(billeder fra samme område)
Tonsil fix. 24h/NBFCD138 clone B-A38PT, High (3-1) 95ºC / 30 minPT, High (3-1) 95ºC / 20 minP/E 65ºCP/E 85ºC
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