A primary
assumption generally made in allocating the
tissue for light microscopy (LM), electron
microscopy (EM), and immunofluorescence (IF)
is this:
Only a few glomeruli are required to constitute a
sufficient sample size for EM and IF.
Therefore, if possible, an area of the
biopsy tissue should be identified in which
there are recognizable glomeruli, and a
small sample of that area, 1 to 3 mm in
length, should be sufficient for EM, with a
second similar sample allocated for IF.
All remaining tissue may then be submitted
for LM.
This pattern of tissue allocation minimizes
the amount of tissue required, and increases
the likelihood that maximal information will
be obtained.
See
How Much
Tissue is
Enough?
Two cores of biopsy tissue have been
placed on a saline-moist filter paper
covering the base of a plastic petri
dish. After glomeruli were identified by
examination through the dissecting
microscope, a 1 to 2 mm piece of tissue
containing glomeruli was separated from
the end of each core. One of these small
pieces of tissue will be placed in a
solution of glutaraldehyde for EM, and
the second piece will be placed in
Michel's solution for IF examination.
The two remaining larger cores
will be placed in formalin for LM.
Glomeruli are
approximately 0.1 to 0.2 mm in diameter, a
size just large enough to be visible by the
"naked eye", at the limit of resolution of
the human eye. If glomeruli are filled with
RBC's, as they commonly are at the moment of
removal, glomeruli can ordinarily be readily
seen by simple inspection. However, the
RBC's will ordinarily rapidly "bleed out" of
the tissue sample, and glomerular visibility
will rapidly decrease, particularly if
manipulation of the tissue is required or
performed.
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A hand lens may be used to enhance
visibility, but in ordinary practice it
is difficult/impossible to manipulate
and position the hand lens without
interfering with illumination of the
specimen. In addition, to appropriately
divide a small specimen with a hand lens
in one hand and a razor blade in the
other is at the least difficult, and it
may be dangerous.
I have found that a good quality but rather
inexpensive dissecting microscope, with
20 to 40 X magnification and with both
transmitted and epi-illumination (light
from both below and above the specimen)
reliably permits excellent visualization
and division of the biopsy specimen.
See the contents of the biopsy kit in the figure
below.
Leica Dissecting Microscope,
20-40 X.
Biopsy kit contents, deployed for use.
The three color-coded and labeled bottles contain
formalin, glutaraldehyde, & Michel's
solution.
The petri dish contains a filter paper, moistened
with normal saline from the dropper.
The broken wooden splints are used to manipulate
the tissue, and the half razor blade is used
to divide the specimen.
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