Name: Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells (Video) | JoVE Journal
Uploaded: 2009-07-21T00:00:00.000+00:00
Duration: 8 min 20 s
Description: Learn Shidham's method for preparing cell blocks from cytology specimens with individually scattered cells using AV marker and HistoGel. This technique improves immuno profiling accuracy and special stain evaluation.

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5 minutes

The authors thank Chris Chartrand, HT(ASCP) for demonstrating the section cutting of the cell block with AV marker.

Introduction:
This is a video describing Shidham’s method for cell block preparation from liquid based cytology (LBC) specimen using HistoGelTM (Thermo Scientific) (HG). As compared to other random approaches, the following are the two critical features of this protocol for preparing cell blocks from relatively hypocellular specimens with singly scattered loose cells (1-5).
<ol>
<li>This protocol involves steps to concentrate the cells along the plane parallel to the cutting surface of the cell block.</li>
<li>It also includes a beacon-like dark inclusion of AV-marker (Figure 1b), which serves two of the following purposes:<ol style="list-style-type:lower-alpha;">
<li>To visualize the level at which the cells are concentrated. The area of the cell block with the cells of interest now could be visualized by the histotechnologist when the dark colored beacon is exposed during cutting. This ability to monitor would prevent one from cutting through the level with most of the cells, or not cutting too superficial into the level with highest concentration of sample cells.</li>
<li>To serve as a locator reference point in serial cell block section on different slides. This reference point acts as a beacon to help locate particular cells or groups of cells for evaluation of a coordinate immunoreactivity pattern with the SCIP approach (6,7).</li>
</ol></li>
</ol>
PROTOCOL (Figure 2)
Preparation of the sample.
<ol>
<li>Transfer the residual LBC cervical cytology specimen to a flat bottom glass tube (15mm diameter x 45mm) (Figure 2.1 through 2.4).  Place the glass tube into a larger plastic carrier tube (28 x 85mm) and centrifuge.  Remove the glass bottom tube from the carrier tube and pour off the supernatant.</li>
<li>The glass tube is then capped (to prevent spillage of heating water in the next step) and placed back inside a larger flat bottom carrier plastic tube </li>
<li>The carrier plastic tube containing the glass tube is then capped, placed in a centrifuge (with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube), and spun at 1805 G (3000 rpms, rotor radius- 17cm) for five minutes (Figure 2.5).</li>
<li>The tubes are then removed vertically from the centrifuge and the smaller glass tube is removed with forceps from the larger carrier plastic tube without disturbing the sedimented pellet with cells.</li>
<li>The glass tube with specimen is uncapped and the supernatant is poured off taking care not to disturb the flat layer of sediment cells at the bottom (Figure 2.6).</li>
</ol>
Inclusion of the reference coordinate AV-marker and addition of gel
<ol>
<li>A dark beacon AV-marker (about 2 mm X 2 mm size, flat surfaced, fragment of dark colored, sectionable material) (Figure 3) is added as a signpost to the glass tube (Figure 2.7).</li>
<li>Liquefy an aliquot of HG by melting it in microwave for 10 seconds at medium power.</li>
<li>Add 0.5 ml of molten HG to the tube, mix with the sediment quickly, and recap it (Figure 2.8) (Proceed to the next step quickly without allowing the HG to begin solidifying).</li>
<li>Add about 2.5 ml of warm (45° C) water to the carrier plastic tube (Figure 2.9). </li>
<li>The smaller capped glass tube is placed inside the plastic tube with warm water. (This step is necessary to keep the HG from solidifying during the next steps) (Figure 2.9).</li>
<li>The carrier plastic tube is placed in the centrifuge (with swiveling cups and not fixed angle cups so that the cells fall perpendicularly to the flat bottom of the glass tube), and spun at 1805 G (3000 rpms, rotor radius- 17cm) for five minutes. The purpose of this centrifugation step is to push the AV-marker and to concentrate the cells into a layer closer to the cutting surface of the final paraffin embedded cell block (Figure 1d). </li>
<li>The tubes are then removed gently and vertically from the centrifuge taking care not to disturb the sedimented thin layer with sample cells at the bottom.</li>
<li>The larger plastic tube is uncapped and the smaller glass tube is removed vertically by a forceps without disturbing the sediment layer of specimen cells.</li>
<li>The small glass tube is refrigerated in vertical position for 15 minutes to cool and solidify the HG (Figure 2.11).</li>
</ol>
Removal of the cell block as a button of gel with specimen for final processing
<ol>
<li>The solidified HG disk, with the layer of concentrated/sediment specimen at the bottom is dislodged from the flat bottom glass tube by squirting 10% formalin through a 23 gauge needle with the syringe (Figure 2.12).</li>
<li>The needle is inserted along the side of the tube at the periphery of solidified HG disc with specimen (Figure 2.12).</li>
<li>The needle is rotated along the side of the tube while formalin is slowly pushed through the syringe. This results in the separation of the HG button along with dark colored beacon AV-marker and the concentrated specimen in it from the flat bottom of the glass tube (Figure 2.12).</li>
<li>The cell block (gel button with specimen cells) is then placed in a labeled cassette and submitted for tissue processing to prepare paraffin embedded cell blocks (Figure 2.13).</li>
</ol>
Embedding and cutting of the specimen
<ol>
<li>The disk is embedded in paraffin with the dark beacon marker side down as cutting surface (Figure 1). </li>
<li>The block is sectioned until the dark colored AV-marker as a beacon is exposed and clearly visible.</li>
<li>Three to four micron sections are cut from this level which should contain most of the singly scattered cells from the specimen.</li>
<li>The sections are collected on the glass slide for further staining, immunohistochemical staining, or other tests as indicated. The protocols for these tests including the type of slides to use for mounting the sections may vary. Generally for immunostaining, coated slides are used to prevent floating and loss of sections from the slides during the immunostaining steps.</li>
</ol>
Abbreviations used (in alphabetic order): CISH, chromogenic in-situ hybridization tests; FISH, Fluorescent in-situ hybridization tests; FFPE, formalin-fixed paraffin-embedded; FNA, fine needle aspiration; HG, HistoGel™ (Thermo Scientific); LBC, liquid based cytology; PCR polymerase chain reaction;
 
<img src="/files/ftp_upload/1316/1382fig1.jpg" alt="Paraffin-embedded cell block diagram: structure labeled with paraffin, AV-marker, gel, cell layer details." data-altupdatedat="1787630408000" data-alt="figure 1"> Figure 1. The structure of cell block prepared by Shidham’s protocol.
<img src="/files/ftp_upload/1316/1382fig2.jpg" alt="Cell centrifugation process flowchart with Histogel, AV-marker steps for sediment preparation." data-altupdatedat="1787630408000" data-alt="figure 2"> Figure 2. The summary of different steps for preparing cell block from LBC specimen by Shidham’s protocol.
<img src="/files/ftp_upload/1316/1382fig3.jpg" alt="Preparation process flowchart for AV-marker from banana peels using ink and formalin methodology." data-altupdatedat="1787630408000" data-alt="figure 3"> Figure 3. Preparation of AV-marker from banana peels.
<img src="/files/ftp_upload/1316/1382fig4.jpg" alt="Cell block surface comparison; marker visibility in histological section cutting; diagram analysis." data-altupdatedat="1787630408000" data-alt="figure 4"> Figure 4. Comparison of cell blocks and sections with and without AV-marker.
<img src="/files/ftp_upload/1316/1382fig5.jpg" alt="Histology staining comparison, HE stain, low/high power, with/without AV-marker, microscopy images." data-altupdatedat="1787630408000" data-alt="figure 5"> Figure 5. Comparison of cellularity of sections of cell block with and without AV-marker.

<ol><li>Shidham, V. B., Epple, J. Chapter 14, Appendix I: Collection and processing of effusion fluids for cytopathologic evaluation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShidham%2C+VB+%22Cytopathologic+Diagnosis+of+Serous+Fluids%22">Cytopathologic Diagnosis of Serous Fluids</a>. Shidham, V. B., Atkinson, B. F. , Elsevier. Forthcoming.</li>
<li>Varsegi, G., D'Amore, K., Shidham, V. p16INK4a Immunocytochemistry as an Adjunct to Cervical Cytology - Potential Reflex Testing on Specially Prepared Cellblocks from Residual Liquid Based Cytology (LBC) Specimens. Modern Pathology 22: 98th Annual Meeting of United States and Canadian Academy of Pathology. 2009 Mar 7-13, Boston, Ma, , Abstract 424 97a-97a (2009).</li>
<li>Nigro, K., Tynski, Z., Wasman, J., Abdul-Karim, F., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+cell+block+preparation+methods+for+nongynecologic+ThinPrep+specimens%5BTitle%5D)+AND+%22Diagn+Cytopathol%22%5BJournal%5D)">Comparison of cell block preparation methods for nongynecologic ThinPrep specimens.</a> Diagn Cytopathol. 35, 640-643 (2007).</li>
<li>Saleh, H. A., Hammoud, J., Zakaria, R., Khan, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+Thin-Prep+and+cell+block+preparation+for+the+evaluation+of+Thyroid+epithelial+lesions+on+fine+needle+aspiration+biopsy%5BTitle%5D)+AND+%22CytoJournal%22%5BJournal%5D)">Comparison of Thin-Prep and cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy.</a> CytoJournal. 5, 3-3 (2008).</li>
<li>Kyroudi, A., Paefthimiou, M., Symiakaki, H., Mentzelopoulou, P., Voulgaris, Z., Karakitsos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+diagnostic+accuracy+with+a+cell+block+preparation+from+thin-layer+endometrial+cytology%3A+a+feasibility+study%5BTitle%5D)+AND+%22Acta+Cytol%22%5BJournal%5D)">Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study.</a> Acta Cytol. 50, 63-69 (2006).</li>
<li>Atkinson, B. F. Chapter 5: Immunocytochemistry of effusion fluids: introduction to SCIP approach (Chapter 5. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAtkinson%2C+BF+%22Cytopathologic+Diagnosis+of+Serous+Fluids%22">Cytopathologic Diagnosis of Serous Fluids</a>. Shidham, V. B., Atkinson, B. F. , Elsevier. (2007).</li>
<li>Shidham, V. B. Chapter 15, Appendix II: Immunocytochemistry of effusions processing and commonly used immunomarkers. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShidham%2C+VB+%22Cytopathologic+Diagnosis+of+Serous+Fluids%22">Cytopathologic Diagnosis of Serous Fluids</a>. Shidham, V. B., Atkinson, B. F. , Elsevier. (2007).</li>
</ol>

Cell blocks are a valuable tool for evaluation of various cytology specimens (1). Most importantly, in addition to the architectural details of the specimen, cell blocks allow for evaluation of ancillary studies such as immunocytochemistry, fluorescent/chromogenic in-situ hybridization tests (FISH/CISH) and in-situ PCR. A variety of methods for preparation of cell block are described. However, most of these are suitable for non-gynecologic cytology specimens which contain relatively many cells and with tissue microfragments such as in FNA aspirates and some serous fluids such as effusions (1,3,4,5).
Because cell blocks primarily provide the opportunity for immunocytochemical evaluation, their processing should preferably be similar to that of the formalin-fixed paraffin-embedded (FFPE) tissues. Ultimately the results obtained after immunocytochemical evaluation of cell block sections are compared to those with the published literature performed predominantly on FFPE tissue sections. Any alterations in the protocol potentially compromise and nullify the validity of the results obtained on cell blocks processed through different fixatives and reagent sequences other than that used for routine FFPE. Some commercial techniques may have the drawback of processing through a protocol of exposure to other fixative-reagent exposure.
Various methods of cell block preparation are available for specimens with a significant quantity of sediment and tissue fragments. Principally, the concentrated sediments are supported by some gel or coagulation principle. The maneuverable button is then embedded in paraffin after processing like the surgical pathology specimens/biopsies.
The gels used include gelatin, agar, fibrinogen/plasma-thrombin, and other commercial gels such as HG. The methods of concentration vary from simple pelleting of the sediment by centrifugation to concentration of cells along various types of membranes. Examples include: Milipore, collodin (Celloidin) bags or scraping the cells from the cytology smears on glass slides (1). We also evaluated a variety of gels by trying different combinations of agar and gelatin. None of the combinations achieved the a firm enough consistency to obtain an easily maneuverable disc of solidified gel with embedded cells from the specimen in one piece. HG showed appropriate consistency and in our experience the immunostaining results on HG cell block sections have been excellent.
Liquid based cytology (LBC) specimens for cervicovaginal cytology are generally less cellular than non-gynecologic specimens as mentioned above. In addition, the gynecologic LBC specimens predominantly contain individual scattered exfoliated superficial cells from cervicovaginal mucosa. Due to this, appropriate cellularity within the cell block sections may not be achieved without a special approach. As these singly scattered cellular components in the the block cannot be seen by the histotechnologist during section cutting, the level at which the cells start appearing in the sections cannot be appreciated and may be missed, either by cutting past the level with most cells or not cutting deep enough into the level with highest concentration of sample cells (Figure 4). Shidham&rsquo;s protocol addresses both of these issues using HG as embedding medium and conventional lab equipments (2) (Figure 2).
This protocol involves following two major features (Figure 1):
<ol>
<li>Concentration and alignment of singly scattered cells in a narrow plane adjacent and parallel to the cutting surface of the cell block (Figure 5).</li>
<li>Inclusion of a beacon-like dark AV-marker as a signpost. This is critical for achieving following features:<ol style="list-style-type:lower-alpha;">
<li>Identifying and monitoring the level at which the cells are concentrated in the cell block. The exposure of the dark colored signpost highlights the level at which most of the singly scattered cells in the cell block are expected to be located (Figure 4). This prevents the overcutting (cutting past the level with most cells) or undercutting (not cutting deep enough into the level with highest concentration of sample cells) in to the cell block and allow selection of the sections from the level in the cell block corresponding with highest concentration of cells.</li>
<li>The dark colored signpost in the sections also serves as a reference point to survey and identify exactly the same cells in different serial sections of the cell block on different slides (Figure 4). This is critical while interpreting and evaluating the coordinate properties such immunoprofile of particular cells by the SCIP approach to follow the same cells in different sections (6,7).</li>
</ol></li>
</ol>
Although our protocol is standardized and reported for liquid based cervical cytology specimens, it can also be used to enhance diagnostic yield of many other non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc. In addition the AV marker would facilitate improved application of SCIP approach during immunohistochemical evaluation of cell block sections of these specimens. The embedding medium may be replaced by other reagents with appropriate modifications at relevant steps. HG may be replaced by plasma (Fibrinogen) to be gelled by Thrombin at room temperature (1).

cell block preparation from cytology specimen with predominance of individually scattered cells

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

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.

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Learn Shidham's method for preparing cell blocks from cytology specimens with individually scattered cells using AV marker and HistoGel. This technique improves immuno profiling accuracy and special stain evaluation.

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

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