A major research
activity that we perform for our scientists is Laser Micro Dissection (LMD). Over the
years we have performed LMD on many of our donated tissue samples. We have microdissected breast tissue and
lymph node tissue including both normal and tumor cell types. A good part of
this is performed on OCT embedded tissue.
The first step usually is to prepare H&E section(s) and review the
section/area of interest with the pathologist.
Often times we draw lines on the coverslip to mark the areas of interest
and use as a reference while performing LMD.
This slide will be saved in our file for future reference (see Figure 1).
The next step is
cutting tissue sections with the laser microdissecting microscope. In our
establishment, we use Leica AS LMD (Leica microsystems). Cut tissue sections
are then mounted on PEN foil slides (W. Nuhsbaum Inc.), stained and LMD performed
to cut out the areas of interest.
Many times, both cell types of interest are
represented in the same OCT block (as shown in Figure 1 and 2 above) and often
one of the cell types is much smaller compared to the other. When genomic data for example, is generated
from these cells, there could be potential questions raised about cell
contamination. In order to understand if this was a possibility or not, an
image of the LMD slide and written record of how LMD was performed (that is,
both cell types were obtained from the same slide, or each cell type was
obtained from independent slides during LMD) will provide clear answers about any
potential contamination of cells during the downstream processing into nucleic acids
(DNA/RNA).
Record keeping is therefore
very essential here and you can never have too much detail maintained during
LMD processing. We have always recorded
how many sections we cut from each block and used for LMD, how the cells were
captured within each slide and images of the LMD slide. For example, if we are studying tumor and
adjacent normal tissue from the same donor, we document if we captured both
normal and tumor from separate slides or from the same slide. However, even detailed notes cannot beat the
usefulness of an image of the slide. It is beneficial to capture the H&E image
of the slide and an image of the LMD slide before and after performing LMD. If an imaging system is unavailable, another
option would be to save the PEN foil slide and the original H&E slide. Both of these options will tell you exactly
what areas of the slide were laser microdissected. This exercise may seem
tedious and you may need a lot of storage space in the months or years ahead after
performing multiple LMD, but when you are faced with a research scientist who
has questions regarding the tissue morphology, and who is trying to eliminate
potential cell contamination in the interpretation of research results, you
will be glad you kept all of these different pieces of information!
No comments:
Post a Comment