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The renal biopsy
This document provides guidelines for renal biopsy techniques and analysis. It discusses sample size and location requirements, preferred fixation and staining methods for light microscopy, immunofluorescence, and electron microscopy. Key points covered include obtaining a minimum of 25 glomeruli for focal lesions and 7 for transplants. Juxtamedullary glomeruli are preferred since they are earliest involved in FSGS. Fixation methods and optimal stains for assessing features like cellularity, fibrosis, immune complexes, and basement membranes are also outlined. Categories of active versus fibrosing injury and examples of minimal change disease, membranous nephropathy and FSGS under light and electron microscopy are summarized.
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The renal biopsy
- 1. CORE CURRICULUM INNEPHROLOGY- AJKD Agnes B. Fogo. MDs Francis Kuwait APPROACH TO RENAL BIOPSY
- 2. Sample Size (numberof glomeruli) Focal lesions involving a small number of glomeruli - minimum 25 Membranous glomerulonephritis - single Transplant kidney biopsy diagnoses - minimum 7 For most light microscopic assessment - 8 - 10
- 3. Sample Location (Juxtamedullary vsCortical) Subcapsular cortical samples have overrepresentation of global sclerosis related to aging/hypertension and non-specific scarring. Juxtamedullary glomeruli are the earliest to be involved with segmental sclerosis in focal segmental glomerulosclerosis (FSGS). Preferred location.
- 4. Microscopy preferrences Native renalbiopsies should include Light microscopy (LM), Immunofluorescence microscopy (IF), Electron microscopy (EM). For transplant biopsy LM and IF are considered the standard Repeat biopsies only needing LM in many cases
- 5. Allocation of thebiopsy tissue EM - 1mm LM – largest IF - 3-4 mm
- 6. Fixatives LM- Formalin (10%neutral buffered) Paraformaldehyde Bouin’s or Zenker’s (infrequent) IF- Directly frozen, Transport media such as Michel’s (Tissue is stable at room temperature for mailing to central laboratories in this media within one hour) EM- Glutaraldehyde.
- 7. Handling of Tissue Noforceps, manipulate with thin wooden stick to avoid crush artifact. Avoid touching tissue with a LM or EM fixative-contaminated scalpel or razor blade (this contaminates the tissue for IF).
- 8. Inadequate tissue IF tissuefrozen for IF stains — The remaining frozen tissue may be fixed in formalin and processed for LM. LM tissue fixed on the paraffin block- 1) EM study can be done by processing remaining tissue on paraffin block 2) IF study can sometimes be done satisfactorily in tissue that has not been in paraffin blocks too long
- 9. Staining and processing 1)LM- Dehydrated and placed in paraffin block, and multiple serial sections are obtained and stained. Usual stains are- • Hematoxylin & eosin, • Periodic acid–Schiff (PAS), • Silver methenamine (Jones) • Masson trichrome. Usual processing time – 5 hours
- 10. 2) IF- Tissue forIF is surrounded with OCT compound and frozen, and sections are produced Stains - stained with • Fluorescein-tagged antibodies against IgG, IgA, IgM, • Complements C3 and C1q, and light chain. • Complement product C4d may also be stained (best on frozen tissue, with more technical difficulty in staining on paraffin-block tissue) Usual processing time – 2 hours
- 11. 3) EM- EM tissueis processed and embedded in a plastic, hard media and scout sections (so-called thick sections) are obtained Stain - with toluidine blue Usual processing time – 2 days
- 12. Glomerulus Tubules Interstitium Vasculardisease Assessment Morphological Localisation
- 13. Category of Injury- ActiveVersus Fibrosing Active lesions • Proliferation • Necrosis • Crescents • Edema • Active inflammation ( glomerulitis, tubulitis, vasculitis) Fibrosing •Glomerulosclerosis •Fibrous crescents •Tubular atrophy • Interstitial fibrosis •Vascular sclerosis
- 14. Stains Best assessmentof 1. HE Cellularity and morphology 2 . PAS Basement Membrane Mesangial matrix 3. Masson’s Trichrome Fibrosis
- 15. Stains Best assessmentof 4. Jones Silver Basement Membrane Mesangial matrix 5.Congo red Amyloid 6) MSB Fibrin
- 16. STAINING OF RENALTISSUE COMPONENTS FEATURE HE PAS TRICHROME JONES/GMS Cellularity Excellent Excellent Poor Poor Mesangial M Poor Excellent Variable Excellent Glom. Sclerosis Poor Excellent Excellent Good Immune Cox. Poor Poor Variable Negative Basement M. Poor Excellent Good Excellent Fibrosis Poor Poor Excellent Excellent Vascular hyaline Good Poor Good Negative Thrombi Good Poor Good Variable
- 18. • Green- Epithelialcells a) Visceral- capillary walls i.e PODOCYTES b) Parietal- Bowmans capsule & Proximal tubules • Yellow – Endothelial cells lining capillary lumen • Red – mesangial cells • Blue – mesangial matrix
- 24. Lamina rara externa Laminarara interna Lamina densa Endothelial cell Podocyte
- 25.
- 26. EM Effacement of foot processess Condensationof cytoskeleton to BM
- 27.
- 28. Membranous - LM Thickcapillary wall
- 29.
- 30. Effacement of foot processess Subepithelial immunedeposits BM projection between deposits
- 32. IF- mostly IgGglobal, rarely IgM and IgA
- 33. FSGS – LM Sclerosis Hyalinosis Adhesionto Bowmans capsule
- 34.
- 35. IF – IgMand C3
- 36.