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    • Oct 1, 2020
    • 4 min read

Kidney Biopsy From Needle to Report The Basics

Updated: Jun 7, 2023


DIAGNOSTIC WORKUP




Acknowledgment: Based on GlomCon’s Glomerular Disease Virtual Fellowship seminar by faculty speakers Dr. Benjamin Popp, Dr. Geetika Singh, Dr. Mahesha Vankalakunti, and Mr. John Brealey.




Dr. Rachael Kermond Nephrology Fellow Royal Children’s Hospital Australia




Renal Biopsy: Obtaining the core




Indications


Nephrotic syndrome, acute nephritis, rapidly progressive glomerulonephritis, unexplained AKI, non-nephrotic range proteinuria


Contraindications


Absolute: uncontrolled severe HTN, uncontrolled bleeding


Relative: Solitary native kidney, multiple cysts/masses, small hypoechoic kidneys, active renal infection, skin infection, anatomical anomalies


Technique


Percutaneous needle biopsy under ultrasound guidance to obtain two cores (one formalin fixed for LM and one in saline for IF and EM)

  • Cores examined under dissecting microscope and divided while fresh

  • Core length minimum 10mm and diameter minimum 1.2mm

Complications


Bleeding, infection, damage to surrounding structures, pain, AV fistulas




Renal Biopsy: Processing the core




Macroscopic appearance


Use of dissecting microscopy or standard light microscopy to assist in sample adequacy


Glomeruli can be difficult to visualize due to lack of blood, significant sclerosis, fragmented biopsy with significant blood staining


Adequacy


Diameter

  • Aim 14-16 gauge core for individuals >8 years old, given glomeruli 200-250um

Number

  • Generally 8-10 glomeruli, prefer > 10. Likelihood of detecting pathology dramatically increased with > 20 glomeruli

    • If MCD vs FSGS, 25+ glomeruli including juxtamedullary representation

Region:

  • Need to also consider diagnostic regions

    • Adequate tubulointerstitial parenchyma to assess interstitial fibrosis & tubular atrophy (IFTA)

    • Juxtamedullary in MCD vs FSGS

    • Subcapsular in hypertension-associated glomerulopathy

Inadequacy not meant as criticism but aimed to ensure interpret biopsy result with caution


Processing of Sample:


Ensure accompanied by clinical datasheet: allows the pathologist to provide a more accurate final diagnosis rather than interpret the pattern


Formalin pot for light microscopy

  • Delegation of tissue must occur within minutes to ensure maximum preservation of architecture

  • Generally one whole core

  • Core placed in cassette -> formalin fixed -> processed to remove water and replace with alcohol -> infiltrated with wax -> frozen -> sections taken (up to 21 levels)

    • Sections 2-3 micron (each section has a dedicated stain, dependent on the laboratory)

Fresh tissue for

  • Immunofluorescence

    • Snap frozen


  • Electron microscopy

    • Fixed in glutaraldehyde

    • Generally only need a few glomeruli


Basics of light microscopy




Elements evaluated:


Cells: mononuclear, PMN, Eosinophils


Hypercellularity


Glomerular:

  • Mesangial: IgAN, SLE II, IgM Nephropathy

  • Endocapillary: PIGN, MPGN, SLE class III-IV

Interstitial: Tubulointerstitial nephritis


Spaces: Capillary lumen, Bowman’s space


Matrix: Basement membrane, mesangial matrix


Stains:


Haematoxylin and Eosin (H&E Stain) (A) (B) (E)

  • Allows the first impression of the composition of tissue

  • Hematoxylin stains nuclei blue

  • Eosin stains all components of glomerular and capillary wall pink (Podocyte, endothelial cells, immune deposits)

Periodic acid Schiff stain (PAS stain) (D)

  • Stain allows the assessment of the basement membrane

  • Stains carbohydrates in GBM and mesangial matrix but not other components

  • GBM appears more distinct

  • Can demonstrate nodularity

Jones Methenamine-Silver Stain (C)

  • Stain allows the assessment of the basement membrane

  • Stains carbohydrates that can be oxidized to aldehydes, appear black (argyrophilic)

  • Primarily stains the GBM and areas of sclerosis

  • Can demonstrate spikes, double contours

 Masson’s Trichrome Stain (F)

  • Stains to assess connective tissue and vessels

  • Stains collagen blue, uses three solutions

  • Demonstrates subepithelial immune deposits red/pink

  • Can show areas of fibrosis and sclerosis in red/pink




Special stains

  • Von Kossa Stain: Detects calcification

  • BK virus stain: For SV40

  • Sirius red: Demonstrates fibrosis

  • Congo red: Visualization of amyloidosis



Basics of Immunofluorescence: Processing of core




Standard IF


Receive native tissue (without fixation) in Michel’s transport media or saline


Freeze followed by cryostat sectioning into 3 microns


Staining with human antibody conjugated with a fluorochrome (Direct immunofluorescence)

  • Antibodies labeled with fluorochromes against the following panel

    • Immunoglobulins (IgG, IgA, IgM)

    • Components of complement pathway (C1q, C3c, C4)

    • Kappa and lambda

    • Fibrinogen

    • Albumin

  • Fluorochromes

    • FITC (Fluorescein Iso Thiocyanate)

    • Phycoerythrin

    • Alexa Fluor

Incubate (approximately 1 hour)


Visualize on IF enabled microscopy


Paraffin IF


Advantage of providing a permanent record of fixing but difficult to report due to high background staining of plasma and matrix proteins


Significantly less sensitive for C3 GN, bacterial infection-associated GN, primary membranous nephropathy, anti-GBM nephritis


Unmasking

  • Important for suspected light chain proximal tubulopathy and crystalglobulin induced nephropathy



Basics of Immunofluorescence: Interpretation




Need to comment on the following:


Number of glomeruli present


Number of globally sclerosed glomeruli (Often non-specific staining pattern)


Positivity and Intensity of staining: 0-3+ (or 4+)


Staining pattern: Granular, linear, semi-linear, smudgy, garland, coarse, confluent


Location: Peripheral capillary wall, mesangium, capillary lumen, bowman space, tubular basement membranes, vascular wall, nuclear staining


Segmental trapping of C3 and IgM


Internal controls



Basics of Immunofluorescence: Application


IgA nephropathy/HSP (A) Class IV or diffuse lupus nephritis (B) Membranous nephropathy (C) Post infectious glomerulonephritis (D) C1q nephropathy (E) Diabetic Nephrology (F) Image: Singh G et al. World J Nephrol. 2016



Basics of electron microscopy: Processing




  • Identify glomeruli, then cut thin sections of approximately 100nm with a diamond knife, which are subsequently placed on an electrically conductive copper grid

  • Utilizes transmission electron microscope: Electrons transmitted through section: darker tissue = more electron dense




Basics of electron microscopy: Application


  • Presence and degree of cell proliferation: Mesangial vs endothelial

  • Alteration in cell structure (i.e. podocytes)

  • Necrosis or apoptosis of cells

  • Changes in GBM

  • Localization of immunoglobulin deposits (mesangial, subendothelial, subepithelial)




Basics of electron microscopy: Examples




Mesangial electron-dense deposits Example: IgA nephropathy



Mesangial electron-dense deposits Example: Lupus Nephritis Class II



Abundant epimembranous deposits (with spikes) Example: membranous nephropathy



Abundant epimembranous deposits (with spikes) Example: membranous nephropathy



Subepithelial humps Example: infection-related glomerulonephritis


Subepithelial humps Example: infection-related glomerulonephritis


Subendothelial deposits with tubuloreticular inclusions (interferon footprints) Example: lupus class III or IV


Subendothelial deposits with tubuloreticular inclusions (interferon footprints) Example: lupus class III or IV



Electron microscopy images courtesy of Mr. John Brealey




References:




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