Cell block preparation starts with the transfer of the concentrated specimen cells to a flat bottom glass tube held within a larger tube. The cells are then layered at the bottom by centrifugation before adding the AV marker and molten HistoGel. The addition of warm water to the carrier glass tube ensures that the HistoGel remains liquid.
A subsequent centrifugation pushes the AV marker through the gel and concentrates the cells into a layer close to the cutting surface of the cell block. Once solidified, the gel button can be removed and processed for paraffin embedding. Hi, I am Dr.George Varszegi from the cytology section of the Department of Pathology at the Medical College of Wisconsin in Milwaukee, Wisconsin.
And I'm Dr.Vsam, also from Cytopathology section of Department of Pathology at Medical College of Wisconsin Milwaukee. And today we'd like to show you a procedure for preparing cell blocks from cytology preparations with a preponderance of individually scattered cells. We use this procedure to study the immuno profile of the cells in these individually scattered cells in the cell block sections and also for evaluation of the special stains on these sections.
So let's get started. To begin this procedure, transfer about 0.5 milliliters of the preferably concentrated specimen to a 45 millimeters long flat bottom glass tube with a 15 millimeter diameter. Next, place the glass tube into a large flat bottom plastic carrier tube and centrifuge at 1800 Gs for five minutes at room temperature.
After centrifugation, remove the flat bottom glass tube from the plastic carrier tube and pour off the super natan. Taking care not to disturb the flat layer of cell sediment at the bottom. A dark beacon on Johnny Vena or AV marker is added to the cells as a signpost.
After the cells are embedded, an AV marker serves to identify the level at which the cells are concentrated within the paraffin block. Using a special pipette add a previously prepared AV marker flat surfaced cube to the cells in the tube. Now begin the process of adding the gel liquefy an aliquot of histo gel or HG by melting it in a microwave for 10 seconds at medium power or follow the manufacturer's instructions.
Add 0.5 milliliters of the molten HG to the tube containing the sedimented cells. Quickly mix and recap the tube. Proceed immediately to adding about two and a half milliliters of warm water at 45 degrees Celsius to the carrier plastic tube.
Then insert the CAPAs tube containing the cells, the AV marker, and the HistoGel. Working quickly and using warm water prevents the gel from solidifying during subsequent steps. Next centrifuge to push the AV marker through the gel and to concentrate the cells into a layer which will be closer to the cutting surface of the final paraffin embedded cell block.
Use swiveling cups and not fixed angle cups so that the cells concentrate perpendicularly to the flat bottom of the glass tube and spin it 1800 Gs for five minutes at room temperature. When the centrifugation is complete, remove the tubes gently and vertically from the centrifuge, taking care not to disturb the thin sediment layer of cells at the bottom. Using forceps, remove the smaller glass tube vertically without disturbing the sediment layer of specimen cells.
Finally refrigerate the small glass tube in a vertical position for 15 minutes to cool and solidify the gel. At the end of 15 minutes, proceed to remove the gel containing the cells from the glass tube to dislodge the solidified HG disc, which contains a layer of concentrated specimen at the bottom along with the dark colored AV marker. Use a 23 gauge needle attached to a syringe filled with 10%formin.
Start by inserting the needle along the side of the tube at the periphery of the solidified HG disc. Continue by rotating the needle along the side of the tube while at the same time slowly pushing the Formin solution through the syringe. Observe the separation of the HG button from the flat bottom of the glass tube.
The separated gel button containing the dark colored beacon AV marker and the concentrated specimen is termed a cell block finish by placing the cell block in a labeled cassette and submit it for tissue processing to prepare paraffin embedded cell blocks. Once the cell block is embedded, proceed to cutting the specimen to start cutting the disc of the cell block embedded in paraffin mount the paraffin embedded cell block on the microtome section by course cutting for leveling the surface of the paraffin block and exposing the disc of the cell block until the dark colored AV marker is exposed and clearly visible. Once the dark colored AV marker is exposed, cut three to four micron thick sections.
These sections contain concentrated cells from the original specimen. Collect each section on a glass slide for further staining followed by microscopy. Here at low magnification, we see the impact of including an AV inked banana peel marker in the specimen on cellularity in these h and e stained sections.
One can see in the image on the left that this marker allows for monitoring of the section cutting depth to get the level with good cellularity. The AV marker also allows for the orientation of different cells, in particular sections to be followed in different serial sections on other glass slides with the marker. Absent the cutting depth could not be monitored.
Tissue samples were collected at a sectioning plane with scant cellularity. The degree of cellularity present in the samples is even more evident at high magnification with the AV marker present cutting depth can easily be monitored and one can easily avoid obtaining samples with poor cellularity. We have shown you a method of preparing the cell block from the specimen with loosely scattered fragments and cells.
The dark AV marker, which we added during the processing of the cell block, helps allow us to select these sections with the highest concentration of cells. It also enables us to properly orient these sections on glass slides. It is important for the HistoGel to be in a molten stage while the cells are aligning along the flood pla bottom glass tube, which is the cutting surface.
However, this procedure could be modified for other methods including cold methods such as thrombin, fibrinogen by relevant modifications. So that's it. Thank you for watching and good luck with your procedures.
Thank you.
