CORE CURRICULUM IN NEPHROLOGY- AJKD Agnes B. Fogo. MDs Francis

Kuwait

APPROACH TO RENAL BIOPSY

Sample Size (number of 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

Sample Location (Juxtamedullary vs Cortical)

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.

Microscopy preferrences

Native renal biopsies 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

Allocation of the biopsy tissue

EM - 1mm LM – largest IF - 3-4 mm

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.

Handling of Tissue

No forceps, 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).

Inadequate tissue IF tissue frozen 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

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

2) IF- Tissue for IF 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

3) EM- EM tissue is 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

Glomerulus

Tubules

Interstitium Vascular disease

Assessment

Morphological Localisation

Category of Injury- Active Versus Fibrosing

Active lesions• Proliferation• Necrosis• Crescents• Edema• Active inflammation ( glomerulitis, tubulitis, vasculitis)

Fibrosing• Glomerulosclerosis• Fibrous crescents• Tubular atrophy• Interstitial fibrosis• Vascular sclerosis

Stains Best assessment of

1. HE Cellularity and morphology

2 . PAS Basement Membrane Mesangial matrix

3. Masson’s Trichrome Fibrosis

Stains Best assessment of

4. Jones Silver Basement Membrane Mesangial matrix

5.Congo red Amyloid

6) MSB Fibrin

STAINING OF RENAL TISSUE COMPONENTSFEATURE 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

• Green- Epithelial cells a) Visceral- capillary walls i.e PODOCYTESb) Parietal- Bowmans capsule & Proximal tubules

• Yellow – Endothelial cells lining capillary lumen

• Red – mesangial cells

• Blue – mesangial matrix

Lamina rara externaLamina rara internaLamina densa

Endothelial cell

Podocyte

Minimal change disease- LM

EM

Effacement of foot processess

Condensation of cytoskeleton to BM

Effacement of foot processess

Membranous - LM

Thick capillary wall

EM

Effacement of foot processess

Subepithelial immune deposits

BM projection between deposits

IF- mostly IgG global, rarely IgM and IgA

FSGS – LM

Sclerosis Hyalinosis

Adhesion to Bowmans capsule

FSGS

IF – IgM and C3

Thank You