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Electron Microscopy in Renal Biopsy Interpretation—
When and Why We Still Need It
a report by
Mark Haas, MD, PhD
Professor of Pathology and Director of Renal Pathology and Electron Microscopy, Johns Hopkins University, School of Medicine, Baltimore, Maryland
Transmission electron microscopy (EM) was first used in the interpretation of
medical renal biopsies approximately 50 years ago, and EM studies have
been essential to our understanding of the pathological mechanisms
underlying a wide variety of renal diseases, particularly glomerular diseases.
EM, light microscopy, and immunohistology—immunofluorescence (IF)
and/or immunoperoxidase studies of immunoglobulin (Ig) and complement
deposition—have been routinely employed for decades by renal
pathologists in analyzing native kidney biopsies, and the use of these three
modalities together has become the standard of care for renal biopsy
interpretation in the US.1
Of these three modalities, EM is the most costly and labor-intensive, takes
the most time to complete, and requires the most equipment, space, and
technical expertise to be performed properly. The annual budget for our EM
lab, which processes ~1,000 specimens/year, is over $300,000, which does
not include the salaries of pathologists or the cost (or depreciation of value)
of the electron microscopes themselves. As such, it is not surprising that
in the present healthcare environment—which emphasizes cost
containment—the use of EM in renal biopsy interpretation has been
considerably scaled back or even eliminated at many centers in North
America and Europe. However, is this practice penny wise and pound
foolish? It is the goal of this article to provide some guidance for the
selective use of EM in analyzing renal biopsies, but also to provide concrete
evidence that EM still plays a vital role in diagnostic renal pathology.
Electron Microscopy in Native Renal Biopsies
There are a number of diagnoses that cannot be made with a reasonable
degree of certainty without EM. These diagnoses are listed in Table 1, and
include diseases characterized by:
• structural abnormalities of the glomerular basement membrane (GBM),
such as Alport syndrome and thin GBM nephropathy;
• diseases with immune deposits characterized by an organized
substructure, such as fibrillary glomerulonephritis (GN) and 30–50% of
those cases of cryoglobulinemic GN that lack intracapillary hyaline
thrombi comprising the cryoglobulin;2,3
• certain lysosomal storage diseases;
• forms of GN, e.g. post-infectious GN that is beyond the acute phase,
dense deposit disease, and membranoproliferative GN type III, not
having definitive diagnostic features by light microscopy and/or IF, but
having such features that are shown by EM, e.g. large subepithelial
deposits in ‘notch’ regions underlying mesangial areas in post-infectious
GN (see Figure 1C).
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Current Issues
Notably, these forms of GN include IgA-dominant post-infectious GN, which
is increasingly being recognized as a potential complication of
Staphylococcus infections4 and which beyond the acute phase can be
difficult to distinguish from IgA nephropathy by light microscopy and IF
alone. Table 1 also includes minimal change nephropathy, not so much
because of its characteristic (but not specific) features on EM, but because
some cases of membranous nephropathy—particularly early lesions and late
lesions, where there has been widespread resorption of immune complex
deposits and extensive GBM remodeling—may be indistinguishable from
minimal change nephropathy by light microscopy and even IF.
The diagnoses listed in Table 1 account for no more than 10–15% of our
native kidney biopsy diagnoses, not including biopsies showing incidental
evidence of an old, largely healed, subclinical post-infectious GN, the clinical
significance of which remains unclear.5 There are other instances in which EM
provides valuable diagnostic or prognostic information beyond what is
apparent from histological and IF findings (see Table 2). For example, most
cases of membranous nephropathy do not require EM for diagnosis. Still,
certain EM findings such as mesangial deposits and tubulo-reticular
inclusions (TRIs) raise the possibility of a membranous lesion that is secondary
to a systemic disease, e.g. lupus, mixed connective tissue disease, and
hepatitis B, rather than idiopathic.6–8 Furthermore, a number of studies have
shown that membranous lesions with homogeneous deposits—i.e. all of the
same stage—have a lower rate of progression to end-stage renal disease
(ESRD) and/or a greater likelihood of remission than those with
heterogeneous or asynchronous deposits.9,10
The extent of podocyte foot process effacement may be helpful in
distinguishing cases of primary focal-segmental glomerulosclerosis (FSGS)
Mark Haas, MD, PhD, is Professor of Pathology and Director of
Renal Pathology and Electron Microscopy at Johns Hopkins
University School of Medicine, positions he has held since 2004.
Prior to joining Hopkins in 1999, he served on the faculty of
the Departments of Pathology of Yale University School
of Medicine between 1986 and 1989 and The University of
Chicago between 1989 and 1999. Dr Haas is Vice President
of the Renal Pathology Society (RPS) and will become its
President in 2008. He has served on the Editorial Boards of
Kidney International and the American Journal of Kidney Diseases. His main areas of interest are
glomerular diseases, particularly immunoglobulin A (lgA) nephropathy, and renal transplant
pathology. He received his MD and PhD from Duke University, before completing a residency in
anatomical pathology and fellowship training in renal pathology and physiology at Yale-New
Haven Hospital and Yale University, respectively.
E: mhaas@jhmi.edu
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Table 1: Renal Biopsy Diagnoses Usually Requiring
Electron Microscopy
Alport syndrome
Cryoglobulinemic glomerulonephritisa
Dense deposit disease
Diabetic nephropathy—early morphological changes (GBM thickening)
Fabry’s and other lysosomal storage diseasesb
Fibrillary glomerulonephritis
Focal-segmental glomerulosclerosis—early recurrence in renal allograft
Immunotactoid glomerulopathy
Membranoproliferative glomerulonephritis type IIIc
Membranous nephropathy stages I and IVd
Minimal change nephropathy
Post-infectious glomerulonephritis (except acute form with many glomerular neutrophils)
Thin GBM nephropathy
GBM = glomerular basement membrane.
a. In cases without intracapillary hyaline thrombi (pseudothrombi).
b. Light microscopic changes seen on 1µm-thick sections of tissue embedded in Epon for
electron microscopy (EM) are often considered to be diagnostic for Fabry’s, although EM
provides the definitive diagnosis.
c. Includes both mixed membranous/proliferative (Burkholder21) and disrupted GBM (Strife and
West22) types.
d. In early and advanced membranous lesions immunofluorescence findings may be inconclusive,
and silver stain does not show definitive GBM ‘spikes.’
Figure 1: Microscopic Examination of Biopsy
A. By light microscopy, the biopsy shows mesangial hypercellularity and segmental endocapillary
hypercellularity in all of the glomeruli (hematoxylin-eosin stain, original magnification x200).
B. Immunofluorescence staining for C3, showing glomerular capillary loop and mesangial
staining (fluorescein isothiocyanate-conjugated anti-human C3, original magnification x400).
C. Electron microscopy (EM) showing subepithelial electron-dense deposits (arrows) in ‘notch’
regions, underlying the mesangium between adjacent capillary loops (uranyl acetate and lead
citrate stain, original magnification x3,800). D. EM showing subepithelial deposits partially
incorporated into the glomerular basement membrane. A single tubulo-reticular inclusion
(arrow) is present within an endothelial cell (uranyl acetate and lead citrate stain, original
magnification x6,300).
from those that are secondary to glomerular hypertrophy and
hyperfiltration, such as with obesity or in patients with a single kidney
from birth or following a nephrectomy.11 This is particularly relevant in
instances where the clinical history and light microscopic features (e.g.
enlarged glomeruli, perihilar segmental sclerosis) suggest the latter but
proteinuria is within the nephrotic range. The presence of TRIs in a biopsy
showing collapsing glomerulopathy—collapsing FSGS—strongly suggests
the possibility of HIV-associated nephropathy and signifies a need for
HIV testing in a patient who has not yet been tested or who has previously
20
tested negative. Likewise, thickened GBMs in a patient not known to be
diabetic suggest the possibility of latent or unrecognized diabetes with
relatively early nephropathy, and should be an impetus to determine the
patient’s glucose tolerance and/or hemoglobin A1c level. The presence of
a membranous component is not always recognizable without EM in
biopsies showing diffuse proliferative lupus nephritis, yet may explain
persistent proteinuria after successful treatment of the proliferative GN.
Taking into account biopsies showing the lesions listed in Table 1, as well as
biopsies for which EM provided valuable diagnostic or prognostic information
beyond just the main diagnosis, we found EM to be useful in 98 (42%) of 233
consecutive native renal biopsies analyzed during 1996,12 while Pearson et al.13
found EM to be helpful in 75% of biopsies performed during 1990. A recently
published study from Poland14 confirms that these findings are still relevant. In
this study of 113 biopsies, EM was essential to establish the primary diagnosis
in 31%, and provided important additional information in another 13%.
Electron Microscopy in Renal Transplant Biopsies
It has been the practice in our laboratory, as well as in most others in the
US, to perform EM on biopsies of renal allografts only if there is clinical
evidence of recurrent or de novo glomerular disease. The most common
Performing electron microscopy on renal
allograft biopsies may allow for the early
diagnosis and treatment of humoral
injury to the graft, perhaps before any
irreversible graft injury occurs.
indication for performing EM on an allograft biopsy is to rule out recurrent
FSGS in the early post-transplant period. Patients with early recurrences
of FSGS typically exhibit proteinuria, and their allografts often show
extensive podocyte foot process effacement by EM weeks or even months
before the appearance of diagnostic light microscopic lesions of FSGS.15
The role of EM in the evaluation of renal allograft biopsies may expand
in the near future in light of our increasing understanding of the
important prognostic significance of transplant glomerulopathy (TG) on
graft fibrosis16,17 and the role of anti-donor antibodies in the pathogenesis
of TG.17,18 While TG is typically diagnosed as double contours of the GBM
on light microscopic sections stained with periodic acid-Schiff (PAS)
and/or silver stains to highlight the GBM, investigators in Australia
recently reported endothelial and subendothelial abnormalities that are
sensitive early markers of TG, allowing its diagnosis to be made months
to years before light microscopic lesions of CG become apparent.19
Furthermore, in many instances these EM changes became apparent even
before the appearance of peritubular capillary C4d deposition,19 the latter
indicating the presence of anti-donor antibody though not necessarily
antibody-mediated graft injury.20 As such, performing EM on renal
allograft biopsies may allow for the early diagnosis and treatment of
humoral injury to the graft, perhaps before any irreversible graft injury
occurs, although additional studies will be needed to confirm this.
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Table 2: Cases where Electron Microscopy Provides Valuable
Diagnostic or Prognostic Information
Collapsing glomerulopathy—HIV-associated versus non-HIV-associated forms (TRIs)
Focal-segmental glomerulosclerosis—primary versus secondary forms
Latent or unrecognized diabetic nephropathy
Lupus nephritis—membranous component superimposed on diffuse proliferative
Membranous nephropathy—secondary forms (mesangial deposits, TRIs)
Membranous nephropathy—homogeneous versus heterogeneous deposits
Transplant glomerulopathy—detection of early lesions
TRIS = tubulo-reticular inclusions.
Conclusions—A Case in Point
While the studies cited above make a strong case for the continued use of
EM in renal biopsy evaluation, specific biopsies such as one recently received
in our laboratory might serve to add even greater emphasis to this
conclusion. The patient involved is a 37-year-old-woman with a five-year
history of systemic lupus erythematosus (SLE), with intermittent flares
characterized by pleurisy and pericarditis, but no apparent renal
involvement. She presented with a serum creatinine of 2.5mg/dl (up from a
baseline of 0.8), a new onset of hypertension, and a urinalysis showing 2+
protein and 10–20 red blood cells per high-power field. The patient
underwent a renal biopsy on a Friday afternoon. A light microscopic
examination of the biopsy carried out the next day showed mesangial
hypercellularity and segmental endocapillary hypercellularity in all of the
glomeruli (see Figure 1A). A single glomerulus showed a fibrocellular
crescent (not shown). The patient’s nephrologist was called and was told
that the biopsy showed a diffuse proliferative GN. As a result, the patient’s
daily dose of prednisone was increased, and additional therapy (e.g.
cyclophosphamide, mycophenolate mofetil) was considered.
IF studies performed the following Monday showed strong, diffuse
glomerular capillary loop and mesangial staining for C3 (see Figure 1B), with
mild to moderate staining for IgG and IgM, and weak, segmental staining
for IgA and C1q. As was discussed with the nephrologist, these IF findings
(particularly the relatively mild staining for IgG and the weak C1q staining)
were somewhat atypical for an active, diffuse proliferative lupus nephritis,
and as such the decision was made to defer any additional therapy until
after EM studies were completed. The latter showed findings most
consistent with a subacute post-infectious GN in the early stages of
resolution with segmental, large subepithelial deposits the majority in
‘notch’ regions between the adjacent capillary loops (see arrows, Figure 1C),
and others partially incorporated into the GBM (see Figure 1D). No
subendothelial deposits were noted. While rare TRIs were present (see
arrow, Figure 1D), these can be attributed to the fact that the patient has
SLE, and need not imply lupus nephritis.
The final renal biopsy diagnosis was diffuse proliferative GN, most
likely subacute post-infectious GN. The patient was not given
additional treatment, and within two weeks her serum creatinine had
decreased to 0.9mg/dl with urinalysis showing only trace protein and
blood. Without EM it would not have been possible to make this
diagnosis, and as such the patient might well have been given
unnecessary immunosuppressive therapy.
Electron microscopy provides essential
or helpful information in a substantial
fraction of cases, and whether a biopsy
will fall into the latter fraction is often
not apparent from the clinical history.
Clearly, EM is not needed to accurately interpret all, or even most,
medical renal biopsies. However, EM provides essential or helpful
information in a substantial fraction of cases, and whether a biopsy will
fall into the latter fraction is often not apparent from the clinical history.
As such, it remains advisable that tissue for EM be taken from all medical
renal biopsies, with the decision whether to perform EM being left to the
discretion of the renal pathologist. ■

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