Workflows & Protocols: Laser Microdissection for Pathology and Cancer Research

Report on a Leica LMD user workshop held in Brazil

May 04, 2016

Tumor development results from mutations in our DNA. For their deeper analysis, cancer researchers have to dissect the relevant tissue areas. Here we report the reason why laser microdissection is a perfect tool for this purpose and how this was taught in the course of a workshop held in Brazil at the Federal University of Paraná/UFPR and the Centro de Energia Nuclear na Agricultura/USP (CENA).

With the Leica LMD system pure tumor material can be selected and dissected for downstream analysis to ensure 100% pure starting material without any risk of cross contamination with healthy cells.

Laser microdissection for cancer research

Laser Microdissection was commercially developed* for more focused cancer research and mutation analysis. As some tumors need high purity to reveal their secret of the underlying mutation, a dedicated tool to extract microscopic amounts of pure tumor tissue - without any contaminating healthy cells - for unbiased and unmistakable downstream mutation analysis was re-created: laser capture. This technology evolved quickly and meanwhile conquered many different applications starting from pathology, to separate healthy from disease affected cells, through to neuroscience, developmental research or forensic research etc.

In Brazil, during the Leica LMD workshop series hosted by the Federal University of Paraná/UFPR and the Centro de Energia Nuclear na Agricultura/USP (CENA), oncology and pathology research were addressed besides others.

As a preparation exercise some enthusiastic customer prepared LMD membrane slides with different kinds of tumor samples which were brought to the workshops and tumor material was dissected and collected with the Leica LMD system. This supreme discipline for the upright Leica LMD systems was demonstrated by the Product Manager Dr. Falk Schlaudraff and afterwards successfully applied and succeeded by the attendees who brought the corresponding samples (Fig. 1, 2).

Fig. 1: Workshop attendees during the introduction to the Leica LMD system
Fig. 2: Attendees are running the system on their own fter the introduction.

Tumor dissection is in particular interesting for LMD applications as pure tumor material is obtained for mutation analysis. Some types of cancer require high purity for downstream analysis as otherwise the true underlying mutation might be masked by signals of healthy DNA of surrounding cells which are not affected by the tumor so far. With the Leica LMD system a slide with tumor tissue can be observed and pure tumor material can be selected and dissected for downstream analysis to ensure 100% pure starting material without any risk of cross contamination with healthy cells (Fig. 3, 4).

Furthermore some tumors are heterogeneous: subareas of the tumor can have different mutations. Again the Leica LMD system allows to specifically target different tumor areas and collect those into different collection vessels to get a clear picture of individual subareas of the same tumor. Small metastasis can be dissected providing pure tumor tissue for downstream analysis which is nearly impossible without laser microdissection.

 

 

 

Fig. 3: Exemplary Specimen Overviews of a customer sample with many tumor areas before laser microdissection during the customer workshop.
Fig. 4: Same specimen after tumor areas beeing dissected.

H&E staining for laser microdissection

The most common staining protocol for various tumor sections is H&E (Hematoxylin and Eosin). The staining procedure is as easy as:

  1. Dip the slide in distilled pure water for 30 seconds
  2. Dip the slide in Hematoxylin for 1 minute
  3. Dip the slide in water for 30 seconds
  4. Dip the slide in blueing reagent and incubate for 30 seconds
  5. Dip the slide in Eosin solution and incubate 10 seconds
  6. Dip the slide into 70% ethanol for 30 seconds
  7. Dip the slide into 95% ethanol for 30 seconds
  8. Dip the slide into 100% ethanol for 30 seconds
  9. Air-dry the slide at room temperature

Various applications in Oncology and Cancer research have been published over the last 15 years underlining the importance of this technology for pathology research and precise molecular diagnostics. The ease of use of the Leica LMD system has significantly improved from the early days to a technology which allows any user to operate the system after a brief introduction. Less than 5 clicks or even direct real time live dissection using a touch screen are state of the art and allow to extract tumor areas of any size and shape within a minimum need of time.

*First laser microdissection system was invented 1976 far ahead of its commercial importance and success of this amazing technology got forgotten until its re-establishment around the turn of the millennium.

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