1. Generel pitfalls in diagnostic IHC Søren Nielsen Scheme Manager NordiQC Aalborg Hospital, Denmark

3. IHC - Most common pifalls www.nordiqc.org

4. IHC - Most common pifalls

5. Decalcification Preparation Pre- analytic Analytic Post- analytic Tissue Type, Dimension, Laser resection, De-differentiation Fixation Time, Type, Volume Section Thickness Storage Drying Visualization Sensitivity, Specificity Primary antibody Clone, Dilution Buffer, Time, Temp Development Sensitivity, Localization Interpretation Localization Positive/Negative - cut-off level Quantification Reporting Controlment Pre-treatment The IHC multi-parameter complexity Manual Stainer IHC – Most common pitfalls

6. Decalcification Preparation Pre- analytic Analytic Post- analytic Tissue Type, Dimension, Laser resection, De-differentiation Fixation Time, Type, Volume Section Thickness Storage Drying Visualization Sensitivity, Specificity Primary antibody Clone, Dilution Buffer, Time, Temp Development Sensitivity, Localization Interpretation Localization Positive/Negative - cut-off level Quantification Reporting Controlment Pre-treatment With 3 choices for 5 variables in each phase = > 4 million protocols…. Manual Stainer IHC – Most common pitfalls

9. 6 - 48h 8 - 72h IHC – Most common pitfalls

10. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F Am J Clin Pathol. 2003 Jul;120(1):86-92 “ The minimum formalin fixation time for reliable immunohistochemical ER results is 6 to 8 hours in our laboratory, regardless of the type or size of specimen (core biopsy or resection)”. IHC – Most common pitfalls

11. IHC – Most common pitfalls 13 hours versus 79 hours in 10% NBF (the week-end dilemma…..) 101 breast carcinomas: 99 % Concordance between short fixation and long fixation for ER (SP1) 95 % Concordance between short fixation and long fixation for PR (1E2) 98 % Concordance between short fixation and long fixation for HER2 (A0485)

12. IHC – Most common pitfalls PR (5-10%) PR (30%) SF score 3 PF score 6

13. IHC – Most common pitfalls Breast carcinomas, HER-2 PATHWAY, rmAb 4B5 (CC1 Mild, Ab inc. 20 min. 36 °C, UltraView DAB) + + + + Tumour 4 + + + + Tumour 5 + + + + Tumour 2 3+ + + + + Tumour 3 + + + + Tumour 6 3+ + + + + Tumour 7 + + + + Tumour 8 + + + + Tumour 9 + + + + Tumour 1 168 h. NBF 48 h. NBF 24 h. NBF 4 h. NBF Internal IHC validation

14. IHC – Most common pitfalls 4 h 48 h 24 h 168 h Breast carcinoma 3+, HER-2 PATHWAY, rmAb 4B5

15. IHC – Most common pitfalls 4 h 48 h 24 h 168 h Breast carcinoma 1+, HER-2 PATHWAY, rmAb 4B5

16. IHC – Most common pitfalls HER-2 ISH: Breast carcinomas, Dual SISH CCrb ext, P3. 8 m - - + + Tumour 4 + + + + Tumour 5 + + + + Tumour 2 Amp - - + (+) Tumour 3 + + + + Tumour 6 Amp - + + - Tumour 7 - + + + Tumour 8 poly. - + + + Tumour 9 poly. - + + + Tumour 1 168 h. NBF 48 h. NBF 24 h. NBF 4 h. NBF Internal SISH validation

17. IHC – Most common pitfalls 4 h 48 h 24 h 168 h Breast carcinoma, 1+ Dual SISH CCrb ext, P3. 8 m

18. IHC – Most common pitfalls ” 0” day Fixation Fixed in toto 4 hours - sliced and processed

19. IHC – Most common pitfalls

20. IHC – Most common pitfalls 30 min delay a: HER2 IHC b: HER2 ISH 2 hours delay c: HER2 IHC d: HER2 ISH

21. CD4 – SP35 IHC – Most common pitfalls

22. IHC – Most common pitfalls CD10 – 56C6

23. IHC – Most common pitfalls Prostate – Ki67, rmAb clone 30.9 10 % NBF 24h -> 24h 10 % form. acid 10 % NBF + 10 % form. acid 24h

24. IHC – Most common pitfalls Prostate – PIN cocktail – p504s/CK5/p63 10 % NBF 24h -> 24h 10 % form. acid 10 % NBF + 10 % form. acid 24h

25. 60 °C 1h. HER-2: 3+ 80 °C 16h. HER-2: 1+ IHC – Most common pitfalls

28. Pre-treatment / Epitope retrieval: Defined as an unmasking method for ”re-storing” blocked antigens in formaldehyde fixed tissue The key to an optimal IHC reaction… IHC – Most common pitfalls

29. Optimized temperature-time-pH-buffer system ‘ Heating condition’ = temperature × time: 121 º C/1 min 100 º C/20 min 95 º C/40 min 60 º C/24 h. H eat I nduced E pitope R etrieval Device: Water bath MWO Pressure cooker Pressure cooker & MWO Autoclave Steam Considerations: Efficiency Standardization Tissue damage Performance IHC – Most common pitfalls

30. Domestic Prestige Polar Patent Pascal Milestone Ref. A.J. Balaton et al. Appl. Immunohistochem. 4(4):259-263,1996 MWO Pressure cooker 10 20 IHC – Most common pitfalls

31. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F Am J Clin Pathol. 2003 Jul;120(1):86-92 IHC – Most common pitfalls

32. HIER CC1 stand. 60 min. IHC – Most common pitfalls Ton 168h 10 % NBF HIER CC1 short 8 min. Ton 24h 10 % NBF Ton 6h 10 % NBF CD23 rmAb SP23

33. IHC - most common pitfalls NQC B10 HER-2

35. IHC – Most common pitfalls

36. A: CD20, cl. L26 B: Ki67, cl. MIB1 C: HMB45 (D: MOC31) Modified from: Shi et al. J Histochem Cytochem 1995 43:193-201 6 8 - 9 IHC – Most common pitfalls

37. IHC – Most common pitfalls Tonsil 24 h NBF HIER Pascal PC TE pH 9 Ci pH 6 CD3 PS1 CD19 LE-CD19 PMS2 A16-4

39. TE9 optimum Ci6 optimum TE9 excessive IHC – Most common pitfalls

40. Optimal Excessive IHC – Most common pitfalls CD10

41. IHC completed – wash, mounted IHC completed – dried before mounted IHC – Most common pitfalls Ki67 mAb clone MIB1 – HIER CC1 standard BenchMark Ultra

42. IHC completed – wash, mounted IHC completed – dried before mounted IHC – Most common pitfalls CD79a rmAb clone SP18 – HIER CC1 standard BenchMark Ultra

43. IHC – Most common pitfalls Laminin polyclonal Z0097 HIER ER 2 / TE Proteolysis Pepsin / P1

47. Tonsil PLAP antibody Clone PL8-F6 - Internal validation and protocol set-up IHC – Most common pitfalls

48. Tonsil Prod. PLAP antibody Clone PL8-F6 - Internal validation and protocol set-up IHC – Most common pitfalls

49. MB Ab test 1 clone PL8-F6 1:50 Recommendation 1:50 Own optim. + HIER IHC – Most common pitfalls Placenta Seminoma

50. 1:25 / 100 (mo/po) 1:100 / 400 1:400 / 2000 A None None None B Enzyme 1, 8 min Enzyme 1, 8 min Enzyme 1, 8 min C HIER ER 2 pH 9.0 HIER ER 2 pH 9.0 HIER ER 2 pH 9.0 D HIER ER 1 pH 6.0 HIER ER 1 pH 6.0 HIER ER 1 pH 6.0 (E) ER 1 + Enzyme 1, 4 min ER 1 + Enzyme 1, 4 min ER 1 + Enzyme 1, 4 min (F) Enzyme 1, 4 min + ER 1 Enzyme 1, 4 min + ER 1 Enzyme 1, 4 min + ER 1 Same detection system - sensitive, specific, universal (Refine) Only variable in the laboratory is then the pre-treatment ! Test specimens with different levels of the antigen (none, low, medium, high) Optimally compare more clones before implementation IHC – Most common pitfalls

51. Ton 4h Ton 24h Ton 72h Ton 168h Colon Liver Kidney Pancreas Placenta Lung Prostate Breast Skin Brain MB Ab test 1 IHC – Most common pitfalls Tongue Ton 24h + decalc.

52. Tumor 1 Tumor 2 Tumor 3 Tumor 4 Tumor 5 Tumor 6 Tumor 7 Tumor 8 Tumor 9 Tumor 10 Tumor 11 Tumor 11 Tumor 12 Tumor 13 MB Ab test 2 IHC – Most common pitfalls

53. MLH1 clone ES05, 1:20 (Ref.) MLH1 clone EPR3894, 1:250 IHC – Most common pitfalls MLH1 test – Tonsil fixed 4 h NBF

54. MLH1 clone ES05, 1:20 (Ref.) MLH1 clone EPR3894, 1:250 IHC – Most common pitfalls MLH1 test – Colon ad. carc. 1, MLH1 negative – DNA mutation (PCR)

55. CD138 clone MI15, 1:100 TE CD138 clone 5F7, 1:50 TE IHC – Most common pitfalls CD138 test – Tonsil fixed 24 h NBF

56. CD138 clone MI15, 1:100 TE CD138 clone 5F7, 1:50 TE IHC – Most common pitfalls CD138 test – Plasma cytoma fixed 24 h NBF

57. Ref: rmAb EPR2764Y CM 1:25 32M/CC1S/UV+AMP rmAb SP54 1:100 16M/CC1M/UV IHC – Most common pitfalls CDX2 test

58. Ref: rmAb EPR2764Y CM 1:25 32M/CC1S/UV+AMP rmAb SP54 1:100 16M/CC1M/UV IHC – Most common pitfalls CDX2 test

59. Ref: rmAb EPR2764Y CM 1:25 32M/CC1S/UV+AMP rmAb SP54 1:100 16M/CC1M/UV IHC – Most common pitfalls CDX2 test

60. Ref: rmAb EPR2764Y CM 1:25 32M/CC1S/UV+AMP rmAb SP54 1:100 16M/CC1M/UV IHC – Most common pitfalls CDX2 test

61. Ref: rmAb EPR2764Y CM 1:25 32M/CC1S/UV+AMP rmAb SP54 1:100 16M/CC1M/UV IHC – Most common pitfalls CDX2 test

62. IHC – Most common pitfalls – FN (√) 1A10 CD31 FN FN √ √ √ ( √) FN √ FN FN FN FN Low expressor – (√) 5F7 CD138 – √ SY38 SYP Antigen Clone High expressor Non expressor CD5 CD5/54/F6 √ – CD23 MHM6 √ – CD31 SP54 (√) FN, FP CEA TF-3H8-1 √ FP CGA DAK A3 √ – CK-LMW CAM 5.2 √ – MLH1 EPR3894 √ FP MSH2 EPR3943 √ FP PR SP2 √ FP TTF1 8G7G4/1 √ FP

63. IHC – Most common pitfalls “ IHC-Platform” depending markers” √ √ √ GI191E/A8 BCL6 ? √ FN OCT-207 Oct-2 √ √ FN (3%H2O2) PG-B6p BCL6 √ √ √ SP34 BSAP ? √ √ √ √ √ √ √ √ Autostainer √ √ SP19 CD5 Antigen Clone XT / Ultra Bond-max CD4 1F6, 4B12 FN (3%H2O2) √ CD4 SP35 √ √ CD5 4C7 FP √ CD79a JCB117 Weak √ CD79a SP18 √ √ ASMA 1A4 (√) Weak √ BSAP 24 FN (- Weak) Oct-2 MRQ-2 √ ?

64. IHC – Most common pitfalls mAb clone 123C3: Non-VMS 81% passed VMS 13% passed rmAb clone MRQ-42 VMS 100% passed

65. IHC – Most common pitfalls mAb clone A103: Bond 75% passed VMS 12% passed

66. IHC – Most common pitfalls

67. 1:100, PC TE pH 9, EnV+, AS+ 1:100, CC1 pH 8.5, Ul.W., Ultra IHC – Most common pitfalls SF-1 test – mAb clone N1665 R&D systems

70. IHC – Most common pitfalls Biotin based detection systems should not be used for IHC….!

72. Sensitivity 1:25 1:50 1:150 1:500 Complexity IHC – Most common pitfalls EnV. Fl. UltraView Ult.Vis. ONE Quanto Hi Def. EnV.Fl.+ Refine Super Sens. Ultra Vis. LP UltraView+amp

73. Indirect method: 2-step polymer method EnVision+, Env. Flex UltraView UltraVision-one IHC – Most common pitfalls

75. IHC – Most common pitfalls

76. PAX5 SP34 RTU – 32 min in primary, HIER CC1 standard : 3-step Multimer system (UltraView + Amplification) Tonsil & Hodgkin 2-step Multimer system (UltraView) IHC – Most common pitfalls

77. IHC – Most common pitfalls

78. IHC – Most common pitfalls HIER alkaline buffer + 3-step polymer: 89% sufficient, 67% optimal HIER alkaline buffer + 2-step polymer: 32% sufficient, 13% optimal VMS XT/Ultra IHC platform: 24% suffcient, 0% optimal BOND IHC platform: 77% suffcient, 62% optimal

79. IHC – Most common pitfalls

83. CD23 CSQI: Activated B-cells in mantle z. IHC – Most common pitfalls

84. CDX2 CSQI: Pancreatic duct ep. cells IHC – Most common pitfalls