Genetics
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Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues
Chapters
Summary October 20th, 2019
Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results’ interpretation and integration.
Transcript
The primary advantage of our genotyping protocol is that it minimizes contamination with maternal DNA, which is the main challenge when genotyping formalin-fixed, paraffin-embedded molar tissues. Flow cytometry is a fast and inexpensive method that greatly facilitates the diagnosis of hydatidiform moles. For each formalin-fixed paraffin-embedded, or FFPE, product of conception, prepare a four-micron thick hematoxylin and eosin stain section as detailed in this video for morphological evaluation by microscopy.
Place the FFPE blocks on ice for 15 minutes to facilitate the sectioning. Adjust the microtome to cut sections that are four microns thick for microscopic morphological evaluation. Place the cold block in the microtome and cut one section from each block for H&E staining.
Using forceps, transfer each section to a 45 degrees Celsius water bath. Pick up the section from the water bath with a positively-charged slide previously labeled with the sample identification number using a pencil. Place the slides containing the sections in an oven at 65 degrees Celsius to allow the sections to adhere to the slides.
Keep the slides in the oven for 25 minutes. Perform the H&E staining by submerging the slides into the appropriate staining jars for the correct time period as tabulated in the text protocol. Mount the four-micron sections for morphological analysis with mounting medium.
And cover with glass cover slips. Using the H&E slides and a light microscope, select the FFPE block that has the largest amount of chorionic villi. And if possible, the block that has chorionic villi separate from and not intermingled with maternal tissues.
Adjust the microtome to cut sections that are 10 microns thick for DNA extraction. Cut 10 to 30 sections, depending on the amount of chorionic villi in the block. After picking up the sections with slides as before, place the slides containing the sections in an oven at 65 degrees Celsius to allow the sections to adhere to the slides.
Keep the slides in the oven for 20 minutes. Perform the H&E staining by submerging the slides into the appropriate staining jars for the correct time period as tabulated in the text protocol. Leave the slides under the fume hood for a minimum of three hours in order for the toxic xylene odors to dissipate.
Using forceps, tear a tiny piece of paper out of a moistened paper wipe. Under a light stereo microscope, use the forceps and small pieces of water-moistened paper wipes to scrape off all the unwanted maternal tissues from H&E stains 10-micron thick sections. The key for this step is patience, especially for challenging blocks.
It is also important to have someone else take a look to ensure that no maternal tissues are missed. Now, tear a new piece of paper out of the moistened paper wipes and use it to collect the chorionic villi. Place the pieces of paper wipes with their attached chorionic villi into a labeled 1.5 ml tube.
Minimize the amount of paper wipes used in this step as too much may clog the DNA extraction column and consequently reduce the final amount of collected DNA. Follow the DNA extraction protocol from an FFPE kit to perform DNA extraction. Using a lab spectrophotometer, load 1 microliter of DNA and measure absorbance at 216 nm for quantification.
Load 1 microliter of DNA on a 2%agarose gel and run gel electrophoresis at a voltage of 80 to 100 volts for qualitative evaluation. Based on the DNA evaluation results, choose the volume of DNA to be used in the multiplex short tandem repeat PCR amplification. Aim to use a minimum of 1000 nanograms of DNA.
Proceed to PCR amplification, capillary electrophoresis, and data analysis as described in the text protocol. To choose the ideal FFPE block, use H&E slides on a light microscope to select an FFPE block that has about 50 to 70%of its tissues composed of chorionic villi. For blocks that do not have the ideal amount of chorionic villi, enrich for chorionic villi as the sectioning is performed.
To do so, identify which side of the freshly-cut sections contains more chorionic villi according to its corresponding H&E slide. Then, use a blade to cut off the other half that needs to be discarded in order to enrich for chorionic villi. To perform sectioning of the best possible FFPE block, cut four sections that are 50 microns thick using a microtome.
In the case that an ideal FFPE block is not available, aim to maintain the ratio of chorionic villi to maternal tissue nonetheless. For example, if only 30%of the block is made up of chorionic villi, while the rest has maternal tissues, then remove at least half of the section that contains the maternal tissues, and use more sections to compensate. Place the sections in labeled 15 mL tubes.
For deparaffinization and rehydration, work in a fume hood to add each reagent and wait the appropriate time period as tabulated in the text protocol. Then, remove the reagent by vacuum suction with a Pasteur pipette. Next, add four degrees Celsius citrate solution to the 15 mL tubes.
After placing in an 80 degrees Celsius water bath for two hours, let the solution cool down to room temperature. Remove the citrate solution by vacuum suction. Add six milliliters of 1x PBS, vortex, and wait one to two minutes to allow the tissues to settle to the bottom.
If needed, centrifuge the samples at 1000 RPM for 30 seconds. Remove the 1X PBS by vacuum suction. Now add one mL of preheated pepsin solution and place in a 37 degrees Celsius dry bath for 30 minutes.
Vortex every 10 minutes. Prepare the propidium iodide ribonuclease A solution in the last 10 minutes of this incubation. Add another six mL of 1x PBS, vortex, and wait one to two minutes to allow the tissues to settle to the bottom.
Again, remove the 1x PBS by vacuum suction. Add 550 microliters of the propidium iodide ribonuclease A solution, and place the samples in a 37 degrees Celsius dry bath for 30 minutes. Use forceps to place a five cm by five cm piece of 48 micron filtration mesh in the top part of polystyrene round bottom tubes for use with the flow cytometer.
Filter the solution by pipetting the liquid through the mesh and into the tube. The samples are now ready to be run on the flow cytometer. Keep them wrapped in foil until they are ready to be run.
Finally, perform flow cytometry and data analysis as described in the text protocol. Microsatellite genotyping of a partial hydatidiform mole, along with the DNA of the patient and her partner, reveals that the patient's mole is triploid dispermic. The first 1st marker shows two peaks in the product of conception.
The first peak originates from the mother, since only the mother has a peak of this size. Following the same reasoning, the second peak originates from the father since he shares the same allele. Notice how the second peak is a lot higher than the first, indicating that there are probably two doses of the same paternal allele in that peak.
Maternal contamination is very minimal in this product of conception because it displays a very tiny peak at the position of the second maternal allele. The second marker shows three peaks in the product of conception. Two of these peaks originate from the father, and one from the mother.
The third and fourth markers are similar to the first marker, and also show two alleles coming from the father, and one from the mother. Since all four markers consistently show three alleles, two originating from the father and one from the mother, one can conclude that this product of conception is triploid dispermic and confirms the diagnosis of partial mole. For the interpretation of the genotyping results, it's important to be aware of the level of contamination based on the notes taken during the cleaning steps.
For some cases, we are unable to reach a conclusion following these two methods. In such cases, we use other methods such as FISH, to uncover the true genotypes. Microsatellite genotyping has allowed for accurate molar diagnosis and paved the way to better understand the genotypes that are associated with the various features of hydatidiform moles.
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