Preparation of Formalin-Fixed Paraffin-Embedded Tumor Samples: A Protocol to Preserve Tissue and Cytology Samples from Non-small Cell Lung Cancer (NSCLC)

- To begin, obtain lung tumor specimens as tissue samples or as cytology samples from bronchial washes-- a saline wash containing cells from the inside of the airways. To prepare tissue samples, transfer freshly dissected lung tissue into an embedding cassette. Fix and preserve the tissue with the appropriate concentration of formalin.

To prepare cytology samples, first, treat bronchial washes with DL-dithiothreitol for homogenization. Centrifuge and remove the supernatant. Next, treat with a mucolytic solution to liquefy mucus and separate the cells. Centrifuge to pelletize the cells and fix them with formalin. Treat cells with a suitable cell block preparation reagent to concentrate the sample while working with small pellet volumes.

Transfer the pellet into an embedding cassette. Process both sample types by dehydrating them through a series of graded alcohol baths. Remove alcohol using an appropriate clearing agent followed by wax infiltration. Embed the cassette in liquid paraffin wax and allow it to solidify. This forms a layer around the tissue, holding the specimen in place.

Use a microtome to obtain thinly sliced sections. Place the sections on positively charged glass slides to facilitate adhesion. Dry and stain to observe slides under a microscope. In the following protocol, we demonstrate specimen preparation techniques from lung tumor tissue and cytology samples.

To begin, fix the tumor tissue in 10% neutral buffered formalin in a cassette and embed the cassette in paraffin as outlined in the text protocol. Embed the infiltrated tissue inside a mold that is filled with molten paraffin. Let the tissues stay in the mold until the paraffin has solidified. After this, use a microtome to section the paraffin embedded tissue at a thickness of three micrometers.

Transfer the paraffin ribbon to a positively charged glass microscope slide. Then dry the slide for one hour at 37 degrees Celsius. First, collect the bronchial washings in a preservative solution. Transfer the washings to a 50 milliliter conical tube. Add two grams of DL-dithiothreitol and vortex the tube for 30 minutes. Then centrifuge at 250 times g for five minutes at room temperature.

Remove the supernatant and add 10 milliliters of a mucolytic solution. Shake the sample at medium speed for 20 minutes, and centrifuge at 250 times g for five minutes at room temperature. After this, remove the supernatant and deposit the cell pellet into a collection tube containing 10% neutral buffered formalin.

Add four drops of a cell block preparation agent. Transfer the cell pellet to a cassette. Fix the cell pellet for paraffin embedding and sectioning using the process previously described for preparing tumor tissue samples.