The need of electron microscopy in kidney biopsy diagnosis

1. The need of electron microscopy in kidney biopsy diagnosis Yrjö Collan, MD, Dr. Med.Sci., FRCPath Department of Pathology, University of Turku, Finland, and the International University Program, Turku, Finland

2. Elements of the kidney biopsy study in a histopathology laboratory • Traditional light microscopy: thin section, a number of stains including H&E, PAS, silver methenamine, Congo red, Masson´s trichrome. • Immunofluorescence (IF) or other immunomicroscopy including staining for IgG, IgA, IgM, fibrinogen, C1q, kappa and lambda chains • Electron microscopy, glutaraldehyde fixation, osmium tetroxide treatment, plastic embedding, ultrathin sections, staining of sections, study in an electron microscope

3. Evaluation of a study element • Kidney biopsy diagnosis is a complicated decision process and includes the element of uncertainty • To decrease the influence of uncertainty many methods are used in the study of biopsies and the clinical data are carefully associated with the elements of the biopsy study: the whole picture matters. • Evaluation of an individual element can be done by excluding the element from the decision process, and looking how much the exclusion influences the final diagnosis or uncertainties associated with it

4. The categories of EM contribution to diagnosis • Generally authors have found suitable to divide the potential contribution in 3 categories: • 1. EM is absolutely necessary for final diagnosis • 2. EM has supporting influence to final diagnosis, clarifiyng remaining uncertanties • 3. EM appears redundant, i.e. with no influence on final diagnosis or associated uncertainties • We cannot decide about the category if EM has not been done.

5. Revising the diagnosis by re-embedding in plastic • Collan Y, Klockars M, Heino M: Revision of light-microscopic kidney biopsy diagnosis in glomerular disease. Nephron 20: 24-31, 1978 • 18 paraffin embedded biopsies of 1967, positive LM evidence of glomerular disease in 9/18. After re-embedding in Epon, positive EM evidence of glomerular disease in 18/18 cases. Also 1 micron thick plastic sections of the re-embedded biopsies had higher fraction of positive cases than 4 micron thick paraffin embedded biopsies alone.

6. Conclusion from 1978 Improved resolution improves the reliability of diagnosis Re-embedding in plastic may be helpful Spargo (Human Pathol 6:405, 1975) also gave positive evaluation of the role of EM, Muehrke et al. (Arch Intern Med 124:170, 1969) had been more reserved

7. For thin BM nephropathy re-embedding in plastic is not the best solution • Nasr SH, Markowitz GS, Valeri AM, Yu Z, Chen L, D´Agati VD (Columbia Univ, New York, USA): Thin BM nephropathy (TBMN) cannot be diagnosed reliably in deparaffinized, formalin fixed tissue. Nephrol Dial Transplant 22:1228-1232, 2007 • Shrinkage during the process, reflected in a GBM thickness decrease of about 30%

8. Criteria for diagnosis of thin BM nephropathy • Haas M: Arch Pathol Lab Med 130: 699-706, 2006

9. Conclusion on re-embedding in plastic • To get the best material for EM studies you cannot rely on re-embedding • You have to try to get plastic embedded material for EM already at the early phase of the handling of the kidney biopsy

10. Pearson et al. 1994 • Pearson JM, McWilliam LJ, Coyne JD, Curry A: Value of electron microscopy in the diagnosis of renal disease. J Clin Pathol 47: 126-128, 1994 (GB) • EM valuable in about 75%, essential in about 25%, irrelevant in 25%

11. Haas 1997 • A reevaluation of routine electron microscopy in the examination of native renal biopsies. J Am Soc Nephrol 8: 70-76, 1997 (USA) • EM valuable in about 50% of biopsies • However, of all biopsies, material should be embedded in plastic to solve uncertainties

12. Herrera´s group 1997-2002- • Gu X, Herrera GA: The value of electron microscopy in the diagnosis of IgA nephropathy. Ultrastruct Pathol 26:203-210, 2002 (USA) • Herrera GA: The value of electron microscopy in the diagnosis and clinical management of lupus nephritis. Ultrastruct Pathol 23: 63-77, 1999 (USA) • Herrera GA, Isaac J, Turbat-Herrera EA: Role of electron microscopy in transplant renal biopsies. Ultrastruct Pathol 21:481-498, 1997 (USA) • In general EM is valuable, especially in transplant biopsies

13. Wang et al. 1998 • Wang S, Zhang Y, Zou W: The evaluation of electron microscopy in the pathological diagnosis of renal biopsies. Zhonghua Yi Xue Za Zhi 78: 782-784, 1998 (China) • 777 biopsies, EM considered valuable in 32% (about 1/3 of the biopsies)

14. Collan et al. 2005 • Collan Y, Hirsimäki P, Aho H, Wuorela M, Sundstrom J, Tertti R, Metsarinne K: Value of electron microscopy in kidney biopsy diagnosis. Ultrastruct Pathol29:461-468, 2005 (Finland) 85 biopsies 71 nontransplnts Essential 15.3 % 18.3 Additional info 58.8 % 53.5 No extra value 25.9 % 28.2 - Results in line with those of Pearson et al. (1994)

15. Wagrowska-Danilowicz & Danilowicz 2007 • Wagrowska-Danilowicz M, Danilowicz M: Current position of EM in diagnosis of glomerular disease. Pol J Pathol58:87-92, 2007 • EM essential 35 (31.0 %) • EM important 15 (13.3%) • EM not needed 63 (55.7%) Conclusion: EM of value in 44.3%

16. Mubarak & Kazi 2009 • Mubarak M, Kazi JI: Role of immunofluorescence and EM in the evaluation of renal biopsies in nephrotic syndrome in a developing country. Ultrastruct Pathol 33: 260-264, 2009 (Pakistan) 200 biopsies useful essential helpful IF 100% 23.5% 71.5% EM 95.5 43.0 51.5

17. Darouich et al. 2010 • Darouich S, Goucha RL, Jaafoura MH, Moussa FB, Zekri S, Maiz HB: Value of electron microscopy in the diagnosis of glomerular diseases. Ultrastruct Pathol 34:49-61, 2010 (Tunisia) • 52 biopsies, 20 examined with EM, 18 primary disease, 2 transplants. EM essential of helpful in 60%.

18. Elhefnawy 2011 • Elhefnawy NG: Contribution of electron microscopy to the final diagnosis of renal biopsies in Egyptian patients. Pathol Oncol Res 17: 121-125, 2011 • 120 biopsies for primary diagnosis Hereditary Other EM essential 100% 23.5% useful 41.7% unhelpful 33.0%

19. Conclusions • The importance of EM in kidney biopsy diagnosis has increased rather than decreased. Many studies now find that EM is valuable in about 2/3 of the cases. Differences in evaluation may be associated with the material studied, and local factors, including treatment. • For the best service to the patient guarantee the EM option • Training in EM should also be a part or at least an option in the education of pathologists

20. Pathologists and electron microscopy • Furness PN, Boyd S: Electron microscopy and immunohistochemistry in the assessment of renal biopsy specimens: actual and optimal practice. J Clin Pathol 49:233-237, 1996 • Only 4% never requested EM. On average on 74% of biopsies. Many (40%) would like to do EM more often. But the pressure at work and the distance to the EM unit! • Education and management should both be involved

21. As Haas says it • “Electron microscopy provides essential or helpful information to a substantial fraction of cases, and whether a biopsy will fall into the latter fraction is not apparent from the clinical history” • Haas M: Electron microscopy in renal biopsy interpretation - when and why we still need it. US Nephrology1:19-22, 2007

22. Diseases often needing EM (we are looking for signs of these) • Alport and other hereditary nephropathies Cryoglobulinemic glomerulopathies Dense deposit disease (MPG type II) Early diabetic changes Fabry´s disease Recurrence of focal segmental sclerosis Immunotactoid nephropathy Staging membranous nephropathy Thin GBM nephropathy

23. D´Agati et al. 2005 • D´Agati VD, Jennette JC, Silva FG: Non neoplastic kidney diseases. Atlas of nontumor pathology. Am Registry of Pathology 2005 • EM not questioned • Stress the evaluation of location and texture of dense deposits of various kinds

24. A case • 10-year old boy. Macrocytic anemia, and proteinuria. EM of the kidney biopsy shows a few dense deposits in the GBM. Weak IgG deposition, but no complement deposition in immunofluorescence. • What condition? Not mentioned in many kidney biopsy books.

25. Imerslund-Grasbeck syndrome 1 • Hereditary condition. Starts at birth. • Symptoms like in vitamin B12 deficiency. • Cause: the ileal enterocyte receptor for the B12 and intrinsic factor complex is abnormal and the patients do not absorb vitamin B12. • The abnormal receptor is also found in the kidney, tubules and glomeruli.

26. Imerslund-Grasbeck syndrome 2 • Patients often have proteinuria, but not progressive • The protein abnormality in the glomeruli seems to bind IgG, which is found as weak deposition in immunofluorescence investigation, and also seen in EM as dark deposits either in the GBM or in subendothelial position. • No complement deposition is present.

27. Imerslund-Grasbeck syndrome 3 • The genetic abnormality is found at chromosome 14, in which one of 2 protein genes, which are part of the receptor complex, is abnormal (cubilin gene CUBN or amnionless gene AMN).

28. Imerslund-Grasbeck syndrome 4 • References: • Kidney biopsy findings: Collan Y, Lähdevirta J, Jokinen EJ: Selective Vitamin B12 malabsorption with proteinuria. Renal biopsy study. Nephron 23:297-303, 1979 • General: Grasbeck R: Imerslund-Grasbeck syndrome (selective vitamin B12 malabsorption with proteinuria). Orphanet J Rare Dis: 1:17, 2006