Renal Biopsy Work-Up: A Pathologist's Guide

Nephrol Dial Transplant (2006) 21: Editorial Comments
overlap syndrome and small vessel vasculitis. Bone Marrow
Transplant 2004; 33: 1061–1063
15. Ballen KK. New trends in umbilical cord blood transplantation. Blood 2005; 105: 3786–3792
16. Wils EJ, Cornelissen JJ. Thymopoiesis following allogeneic
stem cell transplantation: new possibilities for improvement.
Blood Rev 2005; 19: 89–98
17. Mezey E, Chandross KJ, Harta G, Maki RA, Mc Kercher SR.
Turning blood into brain: cells bearing neuronal
1157
antigens generated from bone marrow. Science 2000; 290:
1779–1782
18. Gangji V, Toungouz M, Hauzeur JP. Stem cell therapy for
osteonecrosis of the femoral head. Expert Opin Biol Ther 2005;
5: 437–442
19. Le Blanc K, Ringden O. Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell
transplantation. Biol Blood Marrow Transplant 2005; 11:
321–334
Received for publication: 2.7.05
Accepted in revised form: 22.12.05
What you should know about the work-up of a renal biopsy
Kerstin Amann1 and Christian S. Haas2
Departments of 1Pathology and 2Internal Medicine IV, University of Erlangen-Nürnberg, Germany
Keywords: renal biopsy; workup; morphology;
immunohistology; immunofluorescence;
electron microscopy
To obtain relevant clinical information from a renal
biopsy is an interdisciplinary task, requiring close
cooperation between clinician and pathologist. The
better the nephrologist’s understanding, the more
rewarding the information from the pathologist.
Based on our experience, we will discuss some practical
points which are not often known or are poorly handled
by our clinical partners.
Since these points require close interaction between
clinicians and pathologists, some guidelines concerning the procedure and work-up of routine kidney
biopsy have been established by the Renal Pathology
Society [1].
Standard procedures for renal biopsy handling and
processing were established at the European Union
Consensus Meeting, which took place on February 25,
2000 in Vienna, Austria; these procedures cover the
following aspects [1]:
If these points are taken into consideration, there is
an 40–50% chance that the results of the kidney
biopsy will have a direct consequence for patient
management [2–6].
Taking the biopsy
Transferring the material
Correspondence and offprint requests to: Professor Dr med. Kerstin
Amann, Department of Pathology, University of ErlangenNürnberg, Krankenhausstr. 8–10, D-91054 Erlangen, Germany.
Email: [email protected]
Dividing the sample
Preserving the tissue
Cutting the biopsy sample
Staining the biopsy sample
Reporting the finding
Establishing the diagnosis
What size of the biopsy is minimal and
optimal for the pathologist?
The first important question on which the opinions
of the nephrological and pathological communities
are divided concerns the necessary size of the kidney
biopsy. We recommend obtaining two biopsy cylinders
with a minimal length of 1 cm and a diameter of at least
1.2 mm. The quality of a renal biopsy depends on the
size, i.e. the number of glomeruli: it is generally agreed
that 10–15 glomeruli are optimal; very often 6–10
glomeruli are sufficient and in some cases even
one glomerulus is enough to make a diagnosis.
However, as correctly pointed out by Corwin et al. [7]
‘If the percentage of glomerular involvement in
a biopsy is used to determine the severity of a
focal glomerular lesion, a small biopsy sample size
will lead to considerable misclassification of disease
ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please email: [email protected]
Downloaded from https://academic.oup.com/ndt/article-abstract/21/5/1157/1822136 by guest on 19 November 2019
Nephrol Dial Transplant (2006) 21: 1157–1161
doi:10.1093/ndt/gfk037
Advance Access publication 9 January 2006
1158
Nephrol Dial Transplant (2006) 21: Editorial Comments
severity. In addition, a small biopsy sample size will
make the exclusion of focal disease difficult’. Therefore,
the pathologist has to check the size and quality
of the biopsy sample under a light microscope before
he decides how to process the material. One of the
cylinders should be fixed and used for light microscopy
and the second one for immunohistology, electron
microscopy and additional investigations.
What clinical information is indispensable
to the pathologist?
How should a renal biopsy be handled?
This depends very much on whether a local pathologist
is available or whether biopsy samples have to be
mailed. Potential media are isotonic NaCl for fast
local transport of the material—the ideal procedure
which permits all different types of further processing
by the pathologist: cryopreservation of one part of the
material for immunofluorescence, fixation with paraformaldehyde (PFA) or formaldehyde (4%, buffered,
pH 7.2–7.4) and, in parallel, fixation of a small part of
the biopsy in 3% glutaraldehyde (usually for electron
microscopy) (Table 1).
There are two ways to divide and fix a kidney biopsy
sample:
i. The biopsy is put into isotonic saline and sent
directly to the pathologist who divides the cylinder,
if possible, into two or three parts: the larger portion
will be used for light microscopy, the smaller
portion will be used for immunofluorescence and
Table 1. Fixation and subsequent processing of kidney biospies
Light
microscopy
ImmunoImmunohisto- Electron
fluorescence chemistry
microscopy
PFA (30 min)
NaCl
PFA
Routine processing Cryosections Paraffin
(4 h)
(1 day)
(1 day)
Glutaraldehyde
Ultrathin sections
(1 week)
What you should know about how the
pathologist works-up the renal biopsy
After embedding in paraffin, it is recommended to
cut several serial sections, i.e. 8–16 paraffin sections
(2–3 mm thick) per biopsy, which will then be used
for the following light microscopic and immunohistochemical staining procedures [8].
Light microscopy (Figure 1A–D)
Routine stains (paraffin sections)
Haematoxylin and eosin (HE)
Periodic acid–Schiff’s (PAS)
Fibrous tissue stain (i.e. Sirius red, Trichrom,
Ladewig, etc.)
Silver stain
Protein stain
Optional stains
Kossa stain (calcifications)
Congo red stain (amyloid)
It is useful to apply these different stains because the
information obtained is complementary. For first hand
information in routine diagnostic evaluation, the HE
stain is appropriate (Figure 1A). It provides a first
impression of the composition of the tissue (i.e. renal
cortex vs medulla, number of glomeruli, cellular
infiltration, etc.). To analyse the glomerulus, the PAS
stain is most useful, since it delineates in great detail
glomerular cells, mesangial matrix and potential
expansion, as well as potential modifications of the
composition of the matrix, changes of the GBM, i.e.
thickening, irregularities, doubling, rupture and finally
fibrinoid necrosis of the glomerular tuft (Figure 1B).
A further advantage of the PAS stain is that alterations
of the vessels, particularly arterial hyalinosis and
fibrinoid necrosis, are easy to detect. Immune deposits
can best be visualized by protein stains (Figure 1C)
such as the acid fuchsin–Orange G stain (SFOG).
Often immunodeposits can already be seen or suspected by light microscopy, but definite information
requires specific stains or additional investigations,
i.e. immunohistochemistry or electron microscopy.
Downloaded from https://academic.oup.com/ndt/article-abstract/21/5/1157/1822136 by guest on 19 November 2019
To answer this delicate question, we wish to quote
a statement from computer technology: ‘One cannot
feed in garbage and get out fruit juice’. The absence
of clinical information is a sore point in many partnerships between clinicians and pathologists. In an ideal
world, the pathologist obtains information on the
clinical history, recent laboratory values in particular
urine (proteinuria, haematuria, leukocyturia, cylindruria) and serum [urea, creatinine, cholesterol, total
protein, creatine clearance, C3, C4, anti-nuclear antibody (ANA), anti-neutrophil cytoplasmic antibodies
(ANCA), anti-glomerular basement membrane
(GBM), argininosuccinate lyase (ASL)], presence of
diabetes mellitus or hypertension or other systemic
diseases, other parameters of interest (if available) and
current therapy (if any).
another small portion (if available) will be used for
fixation with glutaraldehyde for examination by
electron microscopy (this is optional).
ii. The biopsy is put directly into a fixative (PFA
or formaldehyde) and mailed to the pathologist.
The pathologist will then embed the material
in paraffin. This permits routine stains to be
performed as well as immunohistology by the
indirect method (APAAP or ABC) using the same
material. A small piece of the formalin-fixed cylinder
can also be worked-up for subsequent electron
microscopy.
Nephrol Dial Transplant (2006) 21: Editorial Comments
1159
The main advantage of the silver stain (Figure 1D)
is that it permits the detection of changes of the GBM,
i.e. so-called reduplication or ‘spikes’ which develop
as a result of overshooting glomerular production
of basement membrane material encircling immunodeposits. In order to evaluate the extent of fibrosis in
the glomerulus or tubular interstitium, fibrous tissue
stains such as Trichrom, Sirius red or Ladewig stains
are indispensable [9]. For specific questions, additional
staining techniques are available, for instance the
Congo red stain for visualization of amyloidosis, or
the Kossa stain to detect calcification.
What are immunohistochemistry and
immunofluorescence, and what information
can be obtained from them?
In order to probe the tissue with antibodies,
two techniques are available: immunofluorescence
Downloaded from https://academic.oup.com/ndt/article-abstract/21/5/1157/1822136 by guest on 19 November 2019
Fig. 1. Overview of the different routine stains in nephropathology. (A) HE stain giving an overview of the renal and in particular
glomerular changes, i.e. glomerular hypercellularity, thickening of glomerular basement membrane, lobular aspect of the glomerular tuft
with occlusion of some capillary lumen. (B) PAS stain useful for the detailed analysis of the glomerular structure. Note the thickening and
irregularities of the glomerular basement membrane. (C) Protein stain (SFOG) documenting marked subendothelial and intramembranous
protein deposits (red colour). (D) Silver stain showing irregularities and thickening of the glomerular basement membrane as well as some
spikes (arrrow) in the periphery of some capillary loops. (E) Immunofluorescence using antisera against IgG. Note the linear staining
in membranous glomerulonephritis. (F) Immunohistochemistry using the antiperoxidase method and an antibody against IgG. As
with immunofluorescence, linear positive staining along the glomerular basement membrane is visible in a case of early membranous
glomerulonephritis.
1160
When is electron microscopy necessary?
It is certainly not necessary to perform electron
microscopy on every kidney biopsy but, because
the result of the pathological investigation cannot
be predicted, it is wise to preserve the material in
such a fashion that ultimately electron microscopy
is still possible. Electron microscopy requires particular
fixation and handling of the material. It is therefore
time-consuming and not universally available. If electron microscopy is envisaged at the time of a biopsy,
e.g. for Alport’s disease or thin basement disease, a
portion of the material should be fixed with glutaraldehyde. Alternatively, formalin-fixed tissue can be
used subsequently for electron microscopy.
For some renal diseases, the definite diagnosis
requires electron microscopy, such as Alport’s disease,
thin basement disease, immunotactoid disease, minimal
change nephropathy.
What information is provided
by electron microscopy?
Electron microscopy permits assessment of the
following:
The presence and degree of cell proliferation
(mesangial vs endothelial cell proliferation)
Table 2. Systematic analysis of a kidney biopsy
Glomeruli
Tubulointerstitium
Vessels
Focal–diffuse
Segmental–global
Number and size
Cellularity (which cells?)
Deposits
Mesangial matrix
Fibrinoid necrosis
Crescents
Tubular atrophy and/or dilatation
Necrosis of tubular epithelial cells
Interstitial inflammation (mononuclear
cells, granulocytes, eosinophils)
Interstitial fibrosis
Electron microscopy
Additional features (calcification,
giant cells, etc.)
Wall thickening
Hyalinosis
Fibrinoid necrosis
Inflammation of the vessel wall or
the endothelium (endothelialitis)
Changes in cell structure (i.e. podocyte foot process
fusion or podocyte vacuolization)
Necrosis or apoptosis of cells
Changes of glomerular basement membrane
(i.e. thickening, thinning, splicing, irregularities)
Localization of immunoglobulin deposits (i.e.
mesangial, subendothelial or subepithelial)
In some renal diseases, such as lupus nephritis,
specific morphological changes can be detected by
electron microscopy, e.g. fingerprints or tubuloreticular structures.
How should the pathologist report
the findings in a renal biopsy?
It is useful for communication between the clinician
and the pathologist to adhere to a standard format in
the report. In our opinion, the final report of a kidney
biopsy should include information on:
The adequacy of the specimen (number of glomeruli
and arteries)—(although this will sometimes be
painful for the clinician who performed the biopsy)
A description of the morphological changes in a
systematic fashion for each of the compartments
of interest (glomeruli, tubules, interstititum, vessels,
see Table 2)
The results of immunofluorescence/immunohistochemical studies.
The results of the electron microscopy (this is more
time-consuming and will usually require a separate
report later on)
It is useful to give two different types of diagnoses:
first, a descriptive diagnosis (e.g. mesangioproliferative
glomerulonephritis) and then the final diagnosis
(including the results of immunofluorescence/
immunohistochemical, electron microscopic studies
Downloaded from https://academic.oup.com/ndt/article-abstract/21/5/1157/1822136 by guest on 19 November 2019
(Figure 1E) uses labelled antisera or antibodies
(which require native tissue without fixation) and
immunohistochemistry (which can be done with
formalin-fixed tissue, whilst more aggressive fixatives
destroy the epitopes and preclude immunohistochemical investigations) (Figure 1F).
The
workhorses
for
immunofluorescence/
immunohistochemistry are antisera or monoclonal
antibodies against immunoglobulins (IgA, IgG and
IgM) and components of the classical or alternative
complement pathway (C1q, C3c and C4) as well
as - and -light chains, albumin and fibrinogen,
although for research purposes many others are
available. The pathologist should not only report
whether the reaction is positive, but should also
comment on the pattern of staining, e.g. mesangial vs
capillary staining pattern, linear (or pseudolinear)
vs granular staining. If possible, he should also
describe where the deposits are located, e.g. in a
subendothelial, intramembranous or subepithelial
position.
For specific questions, antibodies against amyloid
subunits (AA or AL amyloid, transthyretin, etc.) or
antibodies against viruses (cytomegalovirus, polyomavirus and adenovirus) are available. In renal transplant
biopsies, immunostaining for the C4d fragment of the
complement pathway has become extremely popular.
Although this is still somewhat controversial, it
appears to be helpful in the diagnosis of acute humoral
rejection [10,11].
Nephrol Dial Transplant (2006) 21: Editorial Comments
Nephrol Dial Transplant (2006) 21: Editorial Comments
as well as clinical information), e.g. IgA glomerulonephritis.
It is hoped that adherence to such a systematic
procedure will improve the results and heighten the
impact of pathology on clinical decision making.
Conflict of interest statement. None declared.
References
5. Andreucci VE, Fuiano G, Stanziale P, Andreucci M. Role of
renal biopsy in the diagnosis and prognosis of acute renal
failure. Kidney Int Suppl 1998; 66: S91–S95
6. Cameron JS. Indications for renal biospy, history of the
procedure, and relationship of the findings to further
investigation and treatment. In: Solez K, Racusen L, Olsen S,
eds. Diagnostic Renal Pathology. Transpath Inc. (http://www.
transpath.com/m-media/DRP.htm)
7. Corwin HL, Schwartz MM, Lewis EJ. The importance
of sample size in the interpretation of the renal biopsy. Am J
Nephrol 1988; 8: 85–89
8. McCarthy GP, Roberts IS. Diagnosis of acute renal allograft
rejection: evaluation of the Banff 97 Guidelines for Slide
Preparation. Transplantation 2002; 73: 1518–1521
9. Amann K. New parameters in kidney biopsy diagnosticmorphometry. Kidney Blood Press Res 2000; 23: 181–182
10. Nickeleit V, Zeiler M, Gudat F, Thiel G, Mihatsch MJ.
Detection of the complement degradation product C4d in renal
allografts: diagnostic and therapeutic implications. J Am Soc
Nephrol 2002; 13: 242–251
11. Böhmig GA, Exner M, Habicht A et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibodydependent graft injury. J Am Soc Nephrol 2002; 13: 1091–1099
Received for publication: 1.12.05
Accepted in revised form: 9.12.05
Nephrol Dial Transplant (2006) 21: 1161–1166
doi:10.1093/ndt/gfl044
Advance Access publication 20 February 2006
Why is homocysteine elevated in renal failure and
what can be expected from homocysteine-lowering?
Coen van Guldener
Department of Internal Medicine, Amphia Hospital, PO Box 9057, 4800 RL Breda, The Netherlands
Keywords: folic acid; homocysteine;
sulphur amino acids
Patients with chronic kidney disease, especially endstage renal disease (ESRD), exhibit many abnormalities
in protein and amino acid metabolism. One of these
alterations involves an increased plasma concentration
of the sulphur-containing amino acid homocysteine.
Hyperhomocysteinaemia has attracted a lot of attention in renal patients, not only because of its close
relationship with renal function, but also because it has
been implicated as an independent cardiovascular risk
Correspondence and offprint requests to: Coen van Guldener,
Department of Internal Medicine, Amphia Hospital, PO Box 9057,
4800 RL Breda, The Netherlands. Email: [email protected]
factor in these patients [1–3], although some recent
studies have found no significant or even an inverse
association between plasma homocysteine level and
cardiovascular events and mortality in ESRD patients
[4–6]. These discordant findings may have been caused
by strong confounders which are associated with low
homocysteine levels and increased mortality, such as
protein energy malnutrition and/or inflammation [7].
Homocysteine and renal function
Plasma homocysteine is strongly correlated with
(estimates of) glomerular filtration rate (GFR).
Hyperhomocysteinaemia, defined as a plasma total
homocysteine level of 12 mmol/l, occurs already at a
GFR of about 60 ml/min and when ESRD has been
ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please email: [email protected]
Downloaded from https://academic.oup.com/ndt/article-abstract/21/5/1157/1822136 by guest on 19 November 2019
1. Regele H, Mougenot B, Brown P et al. Report from Pathology
Consensus Meeting on Renal Biopsy Handling and Processing,
Vienna, February 25, 2000 (http://www.kidney-euract.org/
Rbpathologyconsensus.htm)
2. Thut MP, Uehlinger D, Steiger J, Mihatsch MJ. Renal biopsy:
standard procedure of modern nephrology. Ther Umsch 2002;
59: 110–116
3. Paone DB, Meyer LE. The effect of biopsy on therapy in renal
disease. Arch Intern Med 1981; 141: 1039–1041
4. Fuiano G, Mazza G, Comi N et al. Current indications for
renal biopsy: a questionnaire-based survey. Am J Kidney Dis
2000; 35: 448–457
1161