On behalf of an european concerted action grant on progression of renal
failure (whether interested, please see www.kidney-euract.org), our group
has recently proposed guidelines on renal biopsy handling and processing.
Following, you can find the paper.
Any comments will be greatly appreciated
Maria Pia Rastaldi

Maria Pia Rastaldi, MD
Renal Immunopathology Center
San Carlo Hospital
via Pio II, 3
20153 Milan - Italy
Tel 39-02-40222877
Fax 39-02-40222222
E-mail mp.rastaldi@oscb.sined.net

Consensus Meeting on Kidney Biopsy Handling and Processing

Vienna, February 25th, 2000
Participants:
P.A.J. Brown (Aberdeen), T. Colucci (Bari), L. Gesualdo (Bari), M. Leontsini
(Thessaloniki), B. Mougenot (Paris), M. P. Rastaldi (Milan), H. Regele
(Vienna)

Background

Taking renal biopsies can nowadays be regarded as a safe procedure.
Mortality is extremely low and serious complications requiring surgical
intervention are rare [1-3]. Nonetheless kidney biopsies are done only for
specific indications and no one likes to perform the potentially harmful and
painful procedure repeatedly. Therefore, clinicians and pathologists have to
co-operate tightly in order to ensure an optimal diagnostic yield from any
single biopsy.
In renal pathology, diagnosis is often based on very subtle morphological
changes. To guarantee the detection of all types of diagnostic lesions we
should be able to perform light microscopy (LM) immunohistochemistry (IH)
and electron microscopy (EM) in every single case. There are significant
variations between laboratories regarding the way to process renal biopsies.
A harmonised treatment of renal biopsies would be highly desirable since it
facilitates an exchange of slides or tissue samples for scientific or
quality assessment purposes.
Therefore a group of renal pathologists (all being members of the EU
concerted action grant) met in Vienna/Austria to reach a consensus about a
common protocol for renal biopsy handling to be used within the group. The
main goal was to define a set of procedures to allowing free choice of
various diagnostic and scientific methods, thereby ensuring highest
diagnostic precision for the patient.

Taking the Biopsy

The amount of tissue provided by the clinician is a major determinant for
diagnostic accuracy [4-6]. For the diagnosis of some glomerular diseases
(Membranous Glomerulonephritis, Alport Syndrome) a single glomerulus could
be sufficient. In the vast majority of cases, we need more glomeruli and a
larger area of renal tissue to make a reliable diagnosis. This is especially
true for focal glomerular diseases with a low percentage (5%-25%) of
glomeruli involved (e.g. early crescentic Glomerulonephritis), or if a
stratification of patients has to be done according to the relative number
of diseased glomeruli (e.g. Lupusnephritis WHO III vs. WHO IV).
In both cases, adequate therapy largely depends on the correct histological
diagnosis. Statistical analysis revealed that only biopsies with more than
20 glomeruli (for LM only!) virtually exclude sampling error even in cases
with low frequency of glomerular lesions [5].
To keep the sampling error as low as possible it is recommended to obtain
two biopsies, each containing renal cortical tissue [7]. This not only
provides adequate material for LM but also yields enough tissue for
immunofluorescence (IF) and EM, without sacrificing the diagnostic accuracy
of LM (by diminishing the number of available glomeruli). The size of
needles used for kidney biopsies usually ranges from 14G to 18G. The use of
thicker needles not only has the advantage of providing a larger continuous
area of tissue with more glomeruli but also allows the cutting of more
sections thus further enhancing diagnostic precision.
It is crucial to check the kidney biopsy cores for the presence of cortical
tissue at the time of biopsy to be able to immediately perform additional
punctures in case of inadequate samples [8]. Using a microscope, renal
tissue can easily be discriminated from fat or muscles. It is not difficult
to identify individual glomeruli within a biopsy core and to estimate the
proportion of cortical contained in the sample. This type of examination
does not require a special dissection microscope. A usual lab microscope
with a low power objective (2x-5x) is sufficient to examine a biopsy core,
floating in a drop of saline on a glass slide. For best results, use maximum
light intensity and a nearly closed lower diaphragm, in order to
transilluminate the biopsy core.
Given the importance of yielding adequate material and the ease of
microscopic examination, renal biopsies should not be performed in absence
of a microscope.

Transferring and Dividing the Biopsy

After taking the biopsies and ensuring the presence of cortical tissue the
samples have to be divided and appropriately preserved in order to allow the
application of a wide range of diagnostic methods.
This crucially important work has to be carried out under microscopic
control by a specifically trained person [8] who is familiar with the
clinical question and the diagnostic options of modern pathology. Therefore,
the biopsies should be divided at the pathology department where expert
personnel and the specialised equipment for any type of tissue processing
are always available.

Renal biopsy specimens have to be divided into three pieces in the following
order of priority

1. Light microscopy (largest piece, should contain >20 glomeruli)
2. Immunofluorescence (>5 glomeruli)
3. Electron microscopy (>2glomeruli)

As a general rule approximately 70% of the biopsy should go to LM, 20% go to
IF and 10% are sufficient for EM. The actual size and proportion of samples
will depend on the amount of tissue available and the clinical question. In
certain cases it can be reasonable to omit IF or EM to save material for LM.
If the place where the biopsy is taken and the pathology lab are in the same
hospital, the unfixed biopsy samples should be sent on a wet swab (to
prevent drying) on crushed ice to the pathology department and should be
processed within 20 minutes after taking the biopsy. A sealed container for
the biopsy, immersed into crushed ice permits perfect cooling and prevents
direct contact of tissue with melting water.
If an immediate transfer cannot be accomplished, but the specimens can be
brought to the pathology lab within 6 hours the biopsy has to be divided
before shipment. The samples for LM and EM are fixed immediately (for types
of fixative see below) and one unfixed portion for IF is sent on crushed ice
as described above.
If a biopsy cannot be transferred within 6 hours, the sample has to be
divided into two portions to be fixed for LM and EM.
In rare cases (tiny biopsy, no microscope available) it may be necessary to
put the entire biopsy into the fixative for LM.
Only the fixatives provided by the pathologist who is receiving the kidney
biopsies should be used, because variations in tissue fixation may
negatively affect subsequent staining procedures.

Tissue preservation

Renal biopsies have to be preserved in three different ways, in order to
achieve best results for all three diagnostic methods (LM, IH/IF, EM) that
have to be employed in native kidney histology.

Light Microscopy

Fixation in buffered formalin followed by paraffin embedding is common
practice in most pathology departments. It results in good morphological
quality of sections and allows immmunohistochemical studies on paraffin
embedded specimen. Fixation with Bouin's or Karnovsky's fluid sometimes
followed by embedding in resin is also used for kidney biopsies due to a
superior preservation of morphological details. However, is questionable
whether the advantage of better morphology outweighs the fact that these
fixatives render immunohistochemistry difficult or impossible.
For allograft biopsies and if diagnosis is urgently needed in native kidney
biopsies, a rapid embedding procedure should be available. Fixation time can
be dramatically diminished using heated formalin. However due to the
shortened cycle times for fixation and embedding, only small specimen
(needle biopsies) can be subjected to rapid embedding procedures.

Immunofluorescence

Immunofluoresence is done on sections of frozen unfixed tissue. In order to
achieve good preservation of morphology the tissue has to be snap-frozen.
Snap-freezing is commonly done in two ways.

1. The specimen is immersed into precooled (on dry ice or liquid nitrogen)
2-methyl-butane (Isopentane)
2. The specimen is placed into a small plastic mould (Cryomold, Tissue-Tek;
Sakura Finetek, Inc.) it is covered with OCT compound (OCT compound,
Tissue-Tek; Sakura Finetek, Inc.). The mould is then placed on the surface
(immersion causes bubbling of OCT and might result in loss of samples) of
liquid nitrogen.

The second method of freezing facilitates handling of the biopsy, prevents
drying and allows repeated production of sections from one specimen without
freeze-thaw cycles.

Electron Microscopy

EM studies should be part of the routine work up of kidney biopsies. EM
plays an important diagnostic role in more than 50% of cases and is
essential for a correct diagnosis in up to 25% [9, 10]. Therefore, tissue
samples for EM studies should always be procured. In some cases however,
(after evaluation of LM and IH) it may be possible to omit electron
microscopy without compromising diagnostic accuracy. The most widely used
fixative for EM is a solution containing 0,1%-3% glutaraldehyde and 4%
paraformaldehyde in 0,1M cacodylate buffer. EM studies can also be done from
specimen fixed in 4% buffered formalin (for LM) without a dramatic loss of
morphological details. If necessary even paraffin embedded tissue or frozen
samples can be utilised for EM. Although samples processed in this way
usually display heavy morphological artefacts important information may be
gained under certain circumstances.

The pathology department that is subsequently processing the biopsies should
provide all fixatives. Consistent fixation is particularly important for
successful employment of immunohistochemical studies on paraffin sections.
Fixatives should be send to the clinical departments in appropriately
labelled vials which are also suitable for shipment of fixed specimen back
to the pathology lab.
Adequate clinical information is indispensable for establishing a meaningful
diagnosis. The standard request form created by the EU concerted action
group should be used for transmission of clinical information.

Cutting and Staining the Biopsy

Light Microscopy

Twenty to fifty consecutive serial 2-3um sections should be prepared for use
in histological and immunohistochemical staining procedures. Sections are
mounted on 20 slides each carrying 1-6 sections. Stains employed include
Haematoxylin and Eosin (H&E, slide No. 1), Periodic-Acid-Schiff (PAS, slides
No. 2&8), Silver-Methenamine (Jones stain, slides No. 3&10) and the
trichrome stain Acid-Tuchsin-OrangeG (AFOG, slides No. 9&15). Elastica van
Gieson stain is helpful for the discrimination between rejection related
chronic transplant vasculopathy and non-specific intimal fibrosis [11]. The
remaining unstained slides are available for additional histochemical stains
(Kongo Red etc.) and immunohistochemistry.

Immunohistochemistry

In renal pathology many diagnostic lesions can only be detected and
classified by immunohistochemical examination of renal biopsies. The routine
use of immunohistochemistry is therefore essential for a proper assessment
of kidney biopsies.
Immunohistochemical studies can be performed on 2-3 um paraffin sections
(indirect immunoperoxidase) or 3-4 um frozen sections (immunofluorescence).
Each method has certain advantages but also number of limitations:

  PRO CONTRA

 IH
Better morphological detail
More background staining (paraffin sections)
Permanent staining
Technically difficult, expensive
Same tissue area in IH and LM Limited number of suitable antibodies
High sensitivity

 IF
Little background staining
Poor morphological detail (frozen sections)
Technically simple, fast, low cost
Non-permanent staining
Vast range of suitable antibodies
Different tissue areas in IF and LM
Additional tissue sample required
Expensive storage

In view of the characteristics listed above, every renal pathology lab
should be in grade to perform both of the techniques. The actual choice of
the appropriate method will depend on the clinical question and the amount
of tissue available. The availability of both methods also provides an
excellent reciprocal control in difficult cases. Frozen sections used for
immunofluorescence should also be stained with HE and PAS for histological
examination.
For immunohistochemistry on paraffin sections proteolytic digestion is
needed to unmask antigenic epitopes and to remove plasma proteins. The
optimal procedure for antigen retrieval and background reduction with
proteases has to be determined individually for each antibody. The need of
protease digestion will also depend on duration and type of fixation. For
native kidney biopsies the following antibodies should always be used:
IgG, IgM, IgA, Complement factors (C3, C1q) and fibrin.
Additional antibodies can be necessary for particular cases. Very important
among them: Kappa and Lambda light chains (for detection of
myeloma-associated renal disease) and antibodies against viral antigens (for
use in allograft biopsies).
Detection and visualisation of primary antibody bound to paraffin sections
is usually achieved with an Avidin-Biotin-Peroxidase System.

Report/Diagnosis

A report on kidney biopsies should contain the following information:

1. Adequacy of specimen (Number of glomeruli and arteries)
2. Systematic description of morphological changes in each of the
compartments of interest (glomeruli, vessels, interstitium, tubules).
3. Results of IH/IF
4. Results of EM (usually follows on later)
5. Descriptive diagnosis (using the WHO scheme of classification)

In many cases, it may be necessary to append concluding remarks discussing
potential differential diagnoses and integrating morphological changes with
clinical information.
It will be often very important to estimate the stage (acute/chronic) of a
disease and to judge the reversibility of lesions.

Table 1 summarises the recommendations for biopsy handling described here.


 References

1. Kim D, Kim H, Shin G, Ku S, Ma K, Shin S, Gi H, Lee E, Yim H: A
randomized, prospective, comparative study of manual and automated renal
biopsies. Am J Kidney Dis 32:426-431, 1998
2. Burstein DM, Korbet SM, Schwartz MM: The use of the automatic core biopsy
system in percutaneous renal biopsies: a comparative study. Am J Kidney Dis
22:545-552, 1993
3. Nass K, O'Neill WC: Bedside renal biopsy: ultrasound guidance by the
nephrologist. Am J Kidney Dis 34:955-959, 1999
4. Oberholzer M, Torhorst J, Perret E, Mihatsch MJ: Minimum sample size of
kidney biopsies for semiquantitative and quantitative evaluation. Nephron
34:192-195, 1983
5. Corwin HL, Schwartz MM, Lewis EJ: The importance of sample size in the
interpretation of the renal biopsy. Am J Nephrol 8:85-89, 1988
6. Schwartz MM: The pathologic diagnosis of renal disease, in Heptinstall's
Pathology of the Kidney (vol 1), edited by Jennette JC, Olson JL, Schwartz
MM, Silva FG, 5th ed, Philiadelphia, Lipincott-Raven Publishers, 1998, pp
169-180
7. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, Croker
BP, Demetris AJ, Drachenberg CB, Fogo AB, Furness P, Gaber LW, Gibson IW,
Glotz D, Goldberg JC, Grande J, Halloran PF, Hansen HE, Hartley B, Hayry PJ,
Hill CM, Hoffman EO, Hunsicker LG, Lindblad AS, Yamaguchi Y, et al.: The
Banff 97 working classification of renal allograft pathology. Kidney Int
55:713-723, 1999
8. Pirani CL, Croker BP: Handling and processing of renal biopsy and
nephrectomy specimens, in Renal Pathology: With Clinical and Functional
Correleations (vol 2), edited by Tisher CC, Brenner BM, 2nd ed,
Philiadelphia, J.B. Lippincott Company, 1994, pp 1683-1694
9. Haas M: A reevaluation of routine electron microscopy in the examination
of native renal biopsies. J Am Soc Nephrol 8:70-76, 1997
10. Pearson JM, McWilliam LJ, Coyne JD, Curry A: Value of electron
microscopy in diagnosis of renal disease. J Clin Pathol 47:126-128, 1994
11. Mihatsch MJ, Nickeleit V, Gudat F: Morphologic criteria of chronic renal
allograft rejection. Transplant Proc 31:1295-1297, 1999

----- Original Message -----
From: <TABrecken@aol.com>
To: <Histonet@pathology.swmed.edu>
Sent: Saturday, September 09, 2000 2:33 AM
Subject: Renal biopsies


> I need help.  The pathologists in our lab ask that histotechnologists
perform
> evaluation of adequacy on renal biopsies.  I would like to know who
usually
> does this function in path labs and if there are any
regulations/guidelines
> from NSH or other agencies about who should perform evaluations of renal
> biopsies and what training is needed.  Also, does anyone have any tricks
of
> the trade on evaluating renals.  The radiologists take the sample in
> ultrasound or CT.
>
> Thanks for your help in advance.
>
>