Presentation on Renal Biopsy

1. Renal Biopsy
DR. MD. KABIR ALAM
ASSISTANT PROFESSOR
DEPARTMENT OF PAEDIATRIC NEPHROLOGY
NIKDU, DHAKA.

2. Kidney

3. Background
• The first published report of the use of kidney biopsy in the
diagnosis of medical kidney disease was in 1951.
• Before this, although clinicians recognized clinical syndromes
such as acute nephritis, nephrosis, asymptomatic hematuria,
and chronic kidney failure, how these related to distinct
pathologic processes remained obscure.
• Over the past 50years, renal pathology has evolved gradually
and, by the turn of the century, our ability to diagnose kidney
disease outstripped our knowledge of pathogenesis.

4. Contd.
The kidney biopsy is an effective, valuable and safe procedure
that provides insight into the:
1. Diagnosis of kidney disease,
2. Assess prognosis,
3. Monitor disease progression,
4. Aid in the selection of therapy and
5. Follow the response to treatment.

5. Indication
NS
GN
AKI
Others
CKD,
Transplanted
Kidney

6. Contraindication
Absolute
1. Sepsis,
2.Severe uncontrolled hypertension,
3. A hemorrhagic diathesis,
4. Known or suspected renal
parenchymal infection or malignancy,
5. Solitary ectopic or horseshoe
kidney (except the transplanted
kidney),
6. Patient who is uncooperative.
7. Acute pyelonephritis/perinephritic
abscess
8. Uremia
Relative
Platelet dysfunction often can be
corrected by administration of
desmopressin (DDAVP) which is a
vasopressin analogue that stimulates
platelet aggregation.
Hypertension may represent a
relative contraindication, assuming it
is brought under adequate control
before the biopsy procedure.

7. Pre–kidney biopsy evaluation.
Counseling and consent from parents
History
Bleeding diathesis, allergy to agent
Use of aspirin, NSAIDs, H/O HTN
Physical examination
• Blood pressure
• Biopsy site assessment
Laboratory evaluation
• CBC,BT, CT, PT, blood grouping, HBsAg, Anti-HCV
• Urine R/E, C/S
Ultrasonography of KUB

8. Contd.
Special attention
1. If child on oral anticoagulants, anti-platelet agents or NSAIDs
should be stopped 7 days before biopsy and will restart after
1-2 weeks after biopsy.
2. If child on HD, renal biopsy should performed at least 6
hours after haemodialysis and halt heparin for next 24
hours.
3. A18 gauze needle is used in newborn or infant and in
allograft kidney biopsy. Allograft biopsy approach is from
anterior abdominal wall.

9. Contd.
Biopsy
equipments
• Biopsy gun
• Two test
tubes
• Others
Medicine
• Midazolam
• 2% Lignocaine
• Emergency
medicines
Procedure
• Prone
position,
pillow/sand
bag under
abdomen
• Left kidney
Post biopsy
care &
monitoring

10. Contd.
• The current standard procedure for kidney biopsy involves the
use of real-time ultrasound to guide an automated spring-
loaded biopsy device percutaneously.
• Computed tomography–guided renal biopsy is an alter native
imaging tool, but it exposes the patient to the risks of
radiation.
• Other procedure for renal biopsy are written in book are open
kidney biopsy, laparoscopic kidney biopsy and transjugular
kidney biopsy in special situation.

11. Contd.

12. Post Biopsy Care
• Following kidney biopsy, vitals are checked at frequent
intervals for initial few hours.
• Bed rest is advised for initial 8-10 h.
• In some centers, kidney biopsy is performed as an outpatient
procedure, but in majority of centers, it is an inpatient
procedure.
• Routine post-biopsy ultrasound is not recommended but we
do because sometimes silent perinephric hematoma
occurred.

13. Contd.
Time F/Up
Just arrival at bed Vitals
After 2 hours Vitals +urine output & colour+ local area
After 4 hours Vitals + urine output & colour
After 6 hours Vitals + urine output & colour
After 24 hours Renal biopsy+ dressing off

14. Contd.
Complication Incidence (%)
Microscopic hematuria 100
Macroscopic hematuria 2.7-26.6
Urinary tract obstruction due to blood
clots
0.5-5
Perinephric hamatoma
1. Clinical examination
2. Ultrasound
3. CT scan
1.4
6-70
57-91
Renal arteriovenous fistula 5-18
BT 0.9-4.1
Nephrectomy 0.06
Death 0.02-0.08

15. Contd.
The diagnosis of glomerular disease in renal biopsy specimens
often has at least 5 steps that may occur in different
sequences:
• Preliminary review of available clinical data prior to
specimen examination,
• Light microscopic examination,
• Immunohistologic examination,
• Electron microscopic examination, and
• Integration of all pathologic and clinical data into a final
interpretation and diagnosis.

16. Adequacy of sample
Two biopsy cylinders with a minimal length of 1 cm and a
diameter of at least 1.2 mm.
• Usually 10–15 glomeruli are optimal; very often 6–10
glomeruli are sufficient
• Some cases even one glomerulus is enough(Membranous
nephropathy)
Cortex and medulla Examination
1-2 glomeruli Electron microscope
3-5 glomeruli Immunofluroscence
6 glomeruli Light microscopy(native kidney)
10 glomeruli Light microscopy(renal allograft)

17. Sectioning and staining
• After histologic processing and paraffin embedding, the
tissues are sectioned by microtome.
• Sections are prepared as thin as 3 μm or less for light
microscopy, at least two sections should be placed on each
slide.
• Thicker sections is needed in congo red and
Immunohistochemistry staining.
• There are many acceptable staining protocols; most include
staining alternating slides with hematoxylin and eosin stain
(H&E), periodic acid–Schiff reaction (PAS), silver methenamine
and Massons trichrome, JMS and congo red.

18. Contd.
• Hematoxylin and eosin
(H and E)
• Periodic Schiff (PAS)
• Silver methanamine
stain
• Trichrome
• Others-Congo red for
amyloidosis

19. Contd.

20. Contd.

21. Staining
• The antigens that should be routinely examined include:
1. Immunoglobulins (primarily IgG, IgM and IgA),
2. Complement components (primarily C3, C1q, and C4), fibrin, and
kappa and lambda light chains.
• Additional antibodies may be required in specific circumstances, for
example:
1. Amyloid speciation,
2. Collagen IV alpha chains in hereditary nephritis,
3. IgG subclasses, virus identification,
4. Lymphocyte phenotyping in allografts in suspected cases of PTLD,
5. C4d in allograft biopsies.

22. Contd.
• The tissue for EM may be fixed in 2–3% glutaraldehyde or 1–4%
paraformaldehyde.
• Toluidine blue-stained, 1 μm thick, so-called ‘thick’ sections, are
examined to identify appropriate structures for thin sectioning
and examination with the electron microscope.
• Thick sections are also useful to supplement the paraffin
material (eg a lesion of focal segmental sclerosis may only be
present on the thick section).
• In general, one or two glomeruli are examined
ultrastructurally. Low-, medium- and high-magnification
photographs are taken to include both capillary loops and
mesangial areas.

23. Interpretation
• The evaluation of a kidney biopsy includes examination of
multiple serial sections each with several tissue slices, stained
with a variety of stains to be examined by LM and IHC.
• Careful evaluation of glomeruli, tubules, the interstitium and
the vessels is required.
• The final report should provide a glomerular count with the
number showing global and/or segmental sclerosis.
• In certain situations, other glomerular lesions should be
counted (eg number of crescents, subtyped into cellular,
fibrocellular and fibrous, etc).

24. Contd.
• A description of alterations in tubules, interstitium and vessels
should also be included.
• Two days for LM and IF, and 3–5 days for EM should be
considered routine.
• A nephropathologist must have a thorough understanding of
renal disease as well as good communication with the
nephrologists caring for the patients for correct final
diagnosis.

25. Contd.
Under the microscope, first a low-power screening
examination helps in localizing the defect is in glomerulus,
tubule, and interstitium, and/or blood vessels.
In addition to the site of lesions, the distribution of lesion is
also important from the pathology point of view.
• Diffuse change: Changes occurring in all the glomeruli.
• Focal changes: Changes occurring in few glomeruli only.
• Global changes: Whole glomerulus is involved.
• Segmental changes: Only some part of glomerulus is involved.
The next issue is to categorize whether the lesion is active or
chronic type.

26. A. Light microscopy(LM)
PAS & TRICHROME SILVER
• Basement membrane --red deep blue black
• Mesangial matrix --red deep blue black
• Interstitial collagen --pale blue
• Cell cytoplasm --rust/orange
• Granular Immune complex deposit -/+ bright red orange,
homogenous
• Fibrin- weakly, Bright red orange,
• fibrillar ---- amyloid -------- Light blue orange

27. Contd.

28. Contd.
• Glomeruli Tubules Interstitium Vessels
A. Injury Localization: Glomerular/Vascular/Tubulointerstitial
B. Category of Injury: Active Versus Fibrosing
1. Active lesions a. Proliferation b. Necrosis c. Crescents d. Edema e. Active
inflammation (eg, glomerulitis, tubulitis, vasculitis)
2. Fibrosing a. Glomerulosclerosis b. Fibrous crescents c. Tubular atrophy
d. Interstitial fibrosis e. Vascular sclerosis
C. Types of Lesions
Determination of the nature and pathogenesis of lesions: examination by IF,
EM and LM

29. Contd.
Glomeruli Generalized or focal involvement, size , cellularity, crescents,
Mesangial matrix , necrosis
Pattern of sclerosis (global, segmental)
Basement membrane
Bowman space
Tubules Atrophy ,degeneration, dilation, cellularity, basement
membrane, tubular casts, RBCs, inflammation (tubulitis)
Interstitium Edema, inflammatory exudates(type of cells),
fibrosisInterstitial inflammation Interstitial fibrosis
Blood vessels Afferent, efferent arterioles; interstitial vessels wall
thickening, hyalinosis, fibrinoid necrosis, endothelitis

30. Glomeruli
• Glossary of terms used to describe histologic lesions in glomeruli
• Focal-Involving <50% of glomeruli
• Diffuse-Involving 50% or more of glomeruli
• Segmental-Involving part of a glomerular tuft
• Global-Involving all of a glomerular tuft
• Minimal change: Normal appearance by LM, fusion of podocyte foot processes by
EM
• Mesangial hypercellularity-4 or more nuclei in a peripheral mesangial segment
• Endocapillary hypercellularity-Increased cellularity internal to the GBM composed
of leukocytes, endothelial cells and/or mesangial cells
• Extracapillary hypercellularity-Increased cellularity in Bowman’s space, i.e. > one
layer of parietal or visceral epithelial cells, or monocytes/macrophages.

31. Contd.
• Crescent- Extracapillary hypercellularity collection of cells in Bowman’s
space in response to glomerular damage.
• Fibrinoid necrosis- Lytic destruction of cells and matrix with deposition of
acidophilic fibrin- rich material.
• Mesangiolysis- lysis of mesangial matrix
• Hyaline-Glassy acidophilic extracellular material
• Sclerosis-Increased collagenous extracellular matrix that is expanding the
mesangium, obliterating capillary lumens or forming adhesions to
Bowman’s capsule

32. Contd.
• Membranoproliferative-Combined capillary wall thickening and
endocapillary hypercellularity
• Lobular -Consolidated expansion of segments that are demarcated by
intervening urinary space.
• Humps : Deposits of Ig and complement in a sub- epithelial site
• Spikes : Projections of basement membrane between regular subepithelial
deposit
• Basket weave GBM : The disordered replication of lamina densa of GBM

33. Contd.
No abnormality by light microscopy
1. Glomerular disease with no light
microscopic changes (e.g. minimal
change glomerulopathy, thin
basement membrane nephropathy).
3. Mild or early glomerular disease (e.g.
Class I lupus nephritis, IgA
nephropathy, C1q nephropathy,
Alport syndrome, etc.)
Thick capillary walls without
hypercellularity or mesangial
expansion
1. Membranous glomerulopathy
(primaryor secondary)(>Stage I)
2. Thrombotic microangiopathy with
expanded subendothelial zone
3. Preeclampsia/eclampsia with
endothelial swelling
4. Fibrillary glomerulonephritis with
predominance of capillary wall
deposits

34. Contd.
 Mesangial or endocapillary hypercellularity:
• Focal or diffuse mesangioproliferative glomerulonephritis
• Focal or diffuse (endocapillary) proliferative glomerulonephritis
• Acute (“exudative”) diffuse proliferative postinfectious glomerulonephritis
• Membranoproliferative glomerulonephritis (type I, II or III)
 Extracapillary hypercellularity:
• ANCA crescentic glomerulonephritis (paucity of immunoglobulin by IFM)
• Immune complex crescentic glomerulonephritis ((granular
immunoglobulin by IFM)
• Anti-GBM crescentic glomerulonephritis (linear immunoglobulin by IFM)
• Collapsing variant of focal segmental glomerulosclerosis (including HIV
nephropathy)

35. MCNS

36. PIGN

37. RPGN

38. Lupus nephritis

39. Tubule
Characterized morphologically by destruction/severe injury of the renal
tubular epithelium
• Two major causes are-toxins & ischaemia
Evidence of ATN/injury are-
• Degeneration & necrosis of individual tubular epithelial cells
• Swelling of tubular epithelium(ballooning)
• Detachment of tubular epithelium from underlying BM
• Loss of PAS positive brush border of PCT
• Thinning of tubular epithelium
• Dilatation of tubular Lumina
• Interstitial edema
• Casts ( hyaline, pigmented, eosinophilic, cellular, granular debris)
• Tubular lumen contains sloughed epithelial cells, leukocytes, cellular
debris
• Rupture of tubular BM

40. Contd.
• Lymphocytes or other inflammatory cells on epithelial side of tubular BM
infiltrating the tubular epithelium
Marker of active tubulointerstitial inflammation.
• Hyaline casts
• WBC casts
• Epithelial / granular casts
• RBC casts
• Large hyaline fractured casts
• Myoglobin/hemoglobin casts
Tubules are non functioning & it is no longer capable of regenerating and
resuming function. Tubular BM are thickened & wrinkled.

41. No
abnormality
of tubule
on LM
• Renal vein thrombosis
• Nephrotic syndrome(MCD)
• AGN (acute lupus, APSGN)
• Thrombotic
microangiopathy(HUS)
• Sickle cell disease
• Radiation nephritis
• Amyloid

42. Interstitial
 Interstitial expansion by leukocytes
• Polymorphs(APSGN, drug induced, sepsis)
• Lymphoplasmacytic(chr nephritis, vasculitis, rejection)
• Eosinophils (vasculitis, drug induced, lupus)
• Epitheloid(TB, sarcoidosis, drug induced, malakoplakia)
• Expansion by foam cells
• Hereditary nephritis(Alports syndrome)
• Abundant, prolonged proteinuria or Nephrotic
syndrome (membranous GN)
• Interstitial hemorrhage
• Acute rejection
• Severe GN with rupture of Bowmans capsule
• Malignant HTN
• Vasculitis

43. MPGN

44. Contd.
 Interstitial expansion by neoplastic cells-
• Lymphoma
• Leukemia
• Primary renal ca
• Metastasis
• Crystals & mineral deposits
• Nephrocalcinosis(ca carbonate)
• ARF(ca oxalate)
• Uric acid(gout)
• Cholesterol(glomerular disease with nephrotic syndrome)

45. B. Immunofluresence study(DIF)
Directed at identification of pathogenic Immunoglobulin (Ig) and
complement.
Antibody used routinely-IgG, IgA, IgM, Kappa & lambda light
chains, C3, C4, C1q, fibrinogen
• Glomerular/extraglomerular location, intensity & pattern of
staining
• Glomerular staining catagorized as-mesangial, capilary wall
or both
• Capillary staining –granular, linear, band like

46. Contd.
Linear/ Granular mesangial and capillary wall deposition:
• WHO Class II lupus( full house)
• Finely granular (membranous GN with/without SLE)
• Coarsely granular (MPGN, WHO Class III, or IV lupus)
• Scattered ,coarse granules (poststreptococcal GN)
• Dense deposit disease( ribbonlike,thick C3)
• IgA nephropathy
• C1q nephropathy
• IgM is idiopathic nephrotic syndrome
• Fibrillary GN(IgG)
• Anti GBM disease(IgG, C3)
• Monoclonal Ig deposition disease(mostly kappa chain)
• Primary amyloidosis(usually λ)

47. Contd.
• Dark-field immunofluorescent microscopy is usually graded using a
semiquantitative scale, such as 0, trace, 1+, 2+, and 3+. Some
laboratories divide a positive reaction into 0 to 4+.
• The glomerulus is the usual site of interest, the tubulointerstitium and
the vessels may also react with various antibodies, and a description of
these changes is also required.
• An experienced observer will be familiar with background fluorescence
and the positive control area for each immunoreactant.
• Immunohistochemical staining, most often using diaminobenzidine to
reveal the reactive products, should also be graded and described
fully.

48. IgA nephropathy

49. C. Electron Microscopy
• Electron microscopy, the tool of promise in the 1960s and 1970s, has little
diagnostic utility in the daily practice of anatomic pathology, having been
almost completely replaced by diagnostic immunohistochemical
examination of pathologic tissues.
• The tissue for EM may be fixed in 2-3% glutaraldehyde or 1-4%
paraformaldehyde.
• Tissue can be reprocessed from the paraffin or the frozen block if no
glomeruli are available in the EM sample. However, such reprocessed
tissue will have poor morphologic preservation.
• Toluidine blue-stained 1-μm thick sections are examined to identify
appropriate structures for thin sectioning and examination with the
electron microscope.

50. Contd.
In general, one or two
glomeruli are examined
ultrastructurally.
1. When there is a family
history of renal disease.
2. Hematuria, especially
microscopic, with or
without proteinuria.
3. When there is a
symptomatic proteinuria,
with normal renal
excretory function.
Indication

51. Contd.
• The EM report should include a description of the glomerular
basement membranes, presence or absence of deposits or
infiltrative processes, the status of the foot processes, and changes
in the endothelium.
• Abnormalities of the glomerular basement membranes, such as
wrinkling, folding, collapse, sclerosis, or duplication, thickness
should be described.
• A description of deposits should contain information on location,
density, granularity or fibrillarity, size, and frequency.
• Hypercellularity should be documented, including degree, location,
and cell type, if possible.
• Changes noted in the tubules, the interstitium, or the blood vessels
should also be described.

52. Contd.

53. Contd.
DIF EM
Deposits: IgG, IgM, IgA, C3,C4,C1q,
Fibrin;
Capillary basement membrane:
thickening, thining, wrinkling; diffuse
or focal
Pattern of deposits( granular, linear) Deposits: Site, appearance, abnormal
material ( fibrin,amyloid)
Site: capillary ( subepithelial,
subendothelial, intramembranous),
mesangium
Epithelial cells, foot processes

54. Contd.
• Immunohistochemistry
• IHC detects specific proteins by mono-or polyclonal antibodies raised
against that protein in biopsy. Some of the examples for such proteins are:
Hepatitis B virus and SV40 antigen for BK Polyoma virus infection.
• In-situ Hybridization
• ISH uses labeled cDNA or RNA probes. It localizes specific DNA/RNA
sequence in tissue section which is then quantitated using
autoradiography or fluorescence microscopy. The commonly used ones
are as follows:
• BK virus.
• EB virus probes in the diagnosis of PTLD.
• Pathogenic cytokines such as platelet-derived growth factor, epithelial
growth factor, etc.

55. Final report
• A glomerular count with a statement regarding the number of
obsolescent glomeruli.
• In the case of crescentic glomerulonephritis, the number of
glomeruli with crescents, and of those, the number that are,
for example, cellular, fibrocellular, and fibrous should be
documented.
• A description of the changes seen in the glomerular capillaries
and the mesangium should be given, including information on
alternations in the glomerular basement membranes,
hypercellularity, leukocyte infiltration, matrix expansion, and
the presence of deposits or thrombotic changes, among
others.

56. Contd.
• The other renal compartments should be described as well,
including descriptions of the tubules, the interstitium, and the
blood vessels.
• Occasionally, the only slide containing the pathologic feature
of interest is the toluidine blue–stained, thick section,
produced for EM, which emphasizes the importance of a
careful examination of all available material.
• Finally, detailed communication with the nephrologist or
other clinician caring for the patient leads to an accurate
clinicopathologic correlation and the correct diagnosis.

57. Thanks A Lot