Record Keeping……..Never Too Much: A Lesson Learned in Laser Micro Dissection (LMD)

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!

Using the Smallest Amount Possible

The success of translating bench research to the clinic depends on the availability of good quality biological speciemens. As the demand for well characterized biospecimens increases with increased interest in personalized medicine, Biobanks will face the problem of meeting biospecimen needs for research. The observation today is that sample sizes, for example, from surgical procedures, are decreasing. Factors contributing to this include new advances in diagnostic procedures resulting in much earlier detection of tumors, for example, in cancer. Thus the excised tumor is very small and available only for pathological diagnosis. Also, new techniques such as minimally invasive surgical procedures result in less “left over” material for research.  

These advances are great for medical science. However research activities need to continue so that we can improve and advance our knowledge of disease development and progression. Biobanks therefore will have to design new routine collection methods to feed research needs.  Some of these new methods will include tissue touch imprint preparations, tissue scrapings, and bronchial washes/gastric lavage collections.   These could become sources for isolating DNA and RNA for research. Biobanks will have to assess the potential of incorporating these methods into their routine collection protocols. Specific research/technological platforms may benefit from some or all of these methods while others may not. It will be left to Biobanks to determine what new sample collection methods can be incorporated into their collection protocols when biospecimens from surgical procedures is not an option, and what body fluids can be collected and utilized for nucleic acid extraction other than blood/blood products. These collection methods will not jeopardize patient treatment and “scarce and “small” surgical specimens will now provide biospecimens for research.

  

The image here shows a tissue touch imprint preparation which involves the transfer by direct contact of the superficial cellular content of a fresh tissue (excised from surgery) onto filter paper or a glass slide. Cells from the tissue will adhere to the paper or glass slide. Specific protocols will then be utilized to process the paper or slide for nucleic acids. Touch imprint preparations are widely used by histologists and cytologists.

Safety in the Biorepository

The biorepository is a very valuable asset to Windber Research Institute (WRI), and because of the importance of our samples we take every measure possible to insure safety of samples as well as our personnel. WRI has an active safety committee whose purpose is to bring workers and management together in a cooperative effort to promote safety and health in the workplace.  It is important for each department to be represented and we have several members of the tissue bank on WRI’s safety committee. The committee is formed by lab staff and managers representing at least one member from each department.  The safety committee’s job is to ensure proper lab and workplace safety is being practiced. As members of the committee, we participate in safety inspections of the building and check all areas for safety violations. These inspections are very important and help us to identify existing and potential hazards and recommend corrective action. We check lab equipment, emergency supplies and evacuation plans.

Because operation of a biorepository can potentially expose employees to hazards, it is important for everyone to be involved in training and education activities.  Our biorepository receives annual safety training and participates in periodic educational activities for things such as the safe handling and use of liquid nitrogen, proper personal protective equipment, etc.

Members of the Tissue Bank participate in training for the safe packaging and transport of dangerous goods. The packaging and shipment training helps keep us up to date according to national and international regulations.

Not only is the safety of our staff important, it is critical that we take measures to protect the biospecimens. We have comprehensive standard operating procedures covering everything from physical security and backup power, to monitoring and maintenance of storage equipment and troubleshooting.  We constantly strive to put procedures into place and ensure they are followed with the ultimate goal of protecting our most valuable resource – the biospecimens!