2006 年 1 月 13 日 Journal Reading

1. 2006年1月13日Journal Reading By Jacky Lee

2. Immunohistochemical detection of immunoglobulins and complements in formaldehyde-fixed and paraffin-embedded renal biopsy tissue；an adjunct for diagnosis of glomerulonephritis AKM ROWNAK HOSSAIN CHOWDHURY,1 TAKASHI EHARA,2 MAKOTO HIGUCHI,3 KAZUHIKO HORA3 and HIDEKAZU SHIGEMATSU2 1Department of Histopathology, Shaheed Ziaur Rahman Medical College, Bogra, Bangladesh and Departments of 2histopathology and 3Internal Medical, Shinshu University School of Medicine, Matsumoto, Japan Accepted for publication 16 December 2004 NEPHROLOGY 2005;10, 298–304

3. INTRODUCTION • Microwave treatment（MW）plus protease digestion or trypsin digestion（Try）to demonstrate the deposition of immunoglobulin（Ig）or complements in formaldehyde-fixed and paraffin-embedded（FFPE）renal biopsy tissue • Routine procedure for diagnosis of renal biopsy • Immunofluorescence（IF） • frozen renal tissues • diagnosis of glomerulonephritis • Electron microscopy（EM） • Hematoxylin and Eosin stain & Histochemical stain • fixation of tissues • diagnosis and evaluation of the disease stage

4. INTRODUCTION • No glomerulus in frozen tissue → FFPE tissue • Denatured Antigens → Antigens retrieval • Trypsin digestion • Microwave treatment • Mixed method • Focus comparison： • Microwave treatment plus protease digestion • Trypsin digestion • Conventional immunofluorescence

5. METHODS • Renal Biopsy Samples（Shinshu University School of Medicine） • （7）IgA nephritis • （7）lupus nephritis • （2）mesangial lupus nephritis • （3）membranous lupus nephritis • （2）diffuse lupus nephritis • （7）membranous nephropathy • （2）stage Ⅰ • （2）stage Ⅱ • （3）stage Ⅲ • （3）mesangiocapillary glomerulonephritis（MPGN）

6. METHODS • Tissue Process • Fixed in 30％ formaldehyde • Paraffin embedded • 3 μm section on silane coating slide • Immunological analysis • Direct immunofluorescence • Indirect method • Antigen unmasking methods • Microwave treatment plus protease digestion • Trypsin digestion

7. METHODS • Direct immunofluorescence • Deparaffin （HemoD） • Rehydrate（ethanol） • Wash（PBS） • Antigen unmasking • Wash（PBS） • FITC-labelled Rabbit antihuman IgA, IgG, IgM and C3 antibodies（DAKO）4℃ o/n • Wash（PBS）…

8. METHODS • Indirect mothod • Deparaffin （HemoD） • Rehydrate（ethanol） • Block endogenous peroxidase（1％H2O2 in methanol） • Antigen unmasking • Rabbit antihuman IgA, IgG, C3c and C1q antibodies（DAKO, Kyoto, Japan）4℃ o/n • Goat antirabbit Ig Ab conjugated with peroxidase（DAKO）37℃ 1hr • DAB…

9. METHODS • Antigen unmasking • Microwave treatment（MW-0） • 0.1 mol EDTA pH 8.0 for 10 min • Microwave treatment plus protease digestion • MW ＋0.05％ protease （Sigma, type VII）in PBS for 10 min（MW-10）, 30 min（MW-30）or 60 min（MW-60） • Trypsin digestion • 0.25% trypsin（DIFCO）in PBS pH 7.4 for 30 min（Try-30）, 60 min（Try-60）or 120 min（Try-120）

10. METHODS • Conventional immunofluorescence • Frozen tissue 5 μm section on silane coating slide • Acetone fixed 5 min • FITC-labelled Rabbit antihuman IgA, IgG, IgM and C3 antibodies（DAKO）4℃ o/n • Primary antibodies were omitted as a negative control • Indirect method • No IgM stain • C1q stain → only lupus nephritis • Immunofluorescence • No stain in cases of lupus nephritis

11. RESULTS • （MW-0）did not unmask the antigens • （Try-30）stained one case with low intensity • Protease digestion plus MW treatment • No effective at unmasking the antigens • Protease digestion without MW treatment • Digested more than 30 min • Negative control slides yielded no reaction products or IF

12. RESULTS • Results of IgA Immunofluorescence（IF） • MW treatment plus protease digestion and trypsin digestion → effectively unmasking • MW-10 and MW-30（IgA nephritis, 7/7） • The intensity of IF • FFPE ＜ Frozen • 1 case of IgA nephritis is strong positive • Non-specific staining • Try-60 → 8 cases • Other treatments → 2 or 3 cases

13. RESULTS IgA nephritis, 23 years old woman. Bar, 60 μm DIF in frozen section, 300X IF（MW-30）in FFPE section, 300X

14. RESULTS • Results of IgA Indirect immunostaining • MW-60 and Try-120（IgA nephritis, 7/7） • Try-60（IgA nephritis, 3/7） • The intensity of Indirect immunostaining • 3 cases of IgA nephritis are strong positive • Non-specific staining • No mention • Patterns of IgA deposits • FFPE ＝ Frozen

15. RESULTS IgA nephritis, 23 years old woman. Bar, 60 μm DIF in frozen section, 300X （Try-60）in FFPE section, 300X

16. RESULTS IgA nephritis, 23 years old woman. Bar, 60 μm Patterns of IgA deposits → FFPE ＝ Frozen DIF in frozen section, 300X （Try-60）in FFPE section, 300X IF（MW-30）in FFPE section, 300X

17. RESULTS • Results of IgA immunostaining • Small amounts of IgA deposits • MW treatment → good results • IgA in lupus nephritis • MW-30 and MW-60 → good results • Membranous nephropathy and MPGN • IgA was hardly detected

18. RESULTS IgA nephritis, 17 years old woman. Bar, 45 μm, MW treatment

19. RESULTS

20. RESULTS • Results of IgG Immunofluorescence（IF） • MW treatment plus protease digestion and trypsin digestion → effectively unmasking • Membranous nephropathy, 6/7 • One failure case • EM → subepithelial electron dense deposits • MPGN • 1 case frozen positive → FFPE IgG IF negative

21. RESULTS Membranous nephropathy, 50 years old woman. Bar, 60 μm, Try-60

22. RESULTS • Results of IgG Indirect immunostaining • Membranous nephropathy • MW-10（3/7）；MW-30（5/7） • Lupus nephritis • Try-120（7/7） • MPGN（1/3）；IgA nephritis（0/7） • The intensity of Indirect immunostaining • FFPE ＝ Frozen • Patterns of IgG deposits • FFPE ＝ Frozen

23. RESULTS Diffuse lupus nephritis, 31 years old woman. Bar, 60 μm, MW-30

24. RESULTS • Results of IgG Indirect immunostaining • Non-specific staining • The highest level • MW-10（4） ＞ MW-30 ＞ MW-60 • Try-60（5） • Try-120（3）

25. RESULTS

26. RESULTS • Results of IgM Immunofluorescence（IF） • Frozen section（10/17） • MW-10（7/17） • MW-30（9/17） • MW-60（0/17） • Try-60（8/17） • Try-120（8/17）

27. RESULTS • Results of C3 Immunofluorescence（IF） • Frozen section（13/13） • MW-10（1/13） • MW-30（2/13） • MW-60（0/13） • Try-60（3/13） • Try-120（2/13） • Not well unmasking

28. RESULTS • Results of C3 Indirect immunostaining • MW treatment plus protease digestion • Effective unmask except membranous nephropathy • MW-10（9/20） • MW-30（16/20） • MW-60（14/20） • Try-120（9/20） • Try-120（MPGN, 0/3）

29. RESULTS • Results of C3 Indirect immunostaining • Membranous nephropathy • MW-60（3/6） • Try-120（3/6） • MPGN • Try-120（0/3） • MW-30 & MW-60（3/3） • Lupus nephritis • MW-10 & MW-30（7/7） • MW-60 & Try-120（3/7 or 4/7）

30. RESULTS MPGN, 54 years old woman. Bar, 50 μm, MW-30

31. RESULTS

32. RESULTS • Results of C1q Immunofluorescence（IF） • Only lupus nephritis • Frozen section（7/7） • MW-10 & Try-120（6/7） • MW-30（4/7） • MW-60（3/7）

33. RESULTS • Results of C1q Indirect immunostaining • Only lupus nephritis • MW treatment plus protease digestion • All positive（7/7） • All glomeruli were stained evenly • Trypsin digestion treatment • Not all positive（6/7） • Not stain all of the glomeruli

34. DISCUSSION • Antigens in the glomeruli of FFPE tissue could be unmasked • IgA in IgA nephritis & IgG in lupus nephritis • ☑ MW treatment plus protease digestion • ☑ Trypsin treatment • IgG in membranous nephropathy • IF ＞ Indirect immunostaining

35. DISCUSSION • Detect C3 deposits in FFPE • IF method is no good • Indirect immunostaining • MW-30（80％）vs. Try-120（45％；75％） • Membranous nephropathy • No good（50％） • Detect C1q deposits in FFPE • All good • The intensity of staining • FFPE ≠ Frozen

36. DISCUSSION • MW treatment plus protease digestion • Temperature control unit • MW-0 & MW-10 failed to unmask in FFPE • Trypsin plus MW → steroid hormone receptor • Protease plus MW ↛ antigen in glomeruli • ☒ Over digestion • Trypsin digestion • Try-120 • Non-specific staining • Try ＞ MW

37. DISCUSSION • Detect Ig or C deposits in FFPE • MW plus protease digestion ★★★★★ • Trypsin digestion ★★★★ • C3 in membranous nephropathy ☠☠☠☠☠ • Good method • MW plus protease digestion ＋ Trypsin digestion • 五星級加尚保險

38. DISCUSSION

39. 新年快樂狗年行大運

42. RESULTS