20
Fine-Needle Aspiration Biopsy Techniques
Other Techniques
cell Block Preparation
Cytoblock Cell Block Preparation System, available from Themo
Fisher Scientific; website: http://www.fishersci.com/
Kit includes:
Cytoblock cassettes
Cytoblock reagent 1 (clear fluid)
Cytoblock reagent 2 (colored fluid)
1. Record patient information on the cytoblock.
2. Use samples previously fixed in neutral buffered
formalin.
3. Concentrate the fixed cells by centrifuging the sample
for several minutes. Pour off the excess fluid and
drain the tube on a paper towel.
4. Estimate the amount of sample present. If the total
amount of sample is 2 drops or less, add 4 drops of
reagent 2 to the specimen pellet and mix with a vor-
tex motion.
5. If the sample is more than two drops, divide into
several cell blocks based on 2 drops per block. For
example, if there is 4 drops of sample (enough for
two cytoblocks), add 8 drops of reagent 2 and mix
with a vortex motion.
6. Assemble cytoblock cassettes into cytoclip (cytospin
standard equipment) and keep horizontal. The locat-
ing peg on the back of the cytoblock cassette fits into
the hole in the cytoclip to be properly oriented.
7. Add 3 drops of reagent 1 into the center of the well
in the board insert. Reagent 1 should coat the entire
circumference of the well in the board insert. Use care
to avoid getting any reagent 1 on the top surface of
the board insert.
8. With the backing paper projecting toward the top of
the cytoclip, place a cytofunnel (cytospin equipment)
disposable chamber over the prepared cytoblock and
secure the metal clip holder in the usual manner. (See
cytospin operating instructions.)
9. Place the assembled cytoclip into the cytospin rotor.
10. Place the mixed cell suspension in each cytofunnel.
11. Close the cytospin and set for 5 minutes at 1500 rpm.
Use the
low
acceleration setting. Start the cytospin.
12. When the cytospin stops, remove the cytofunnel
assemblies and place horizontally. Release the clip
and remove the funnels. Removal may require rock-
ing the funnel to the side to separate the funnel
assembly from the underlying board insert. Be certain
the cell button is in the well and has not adhered to
the funnel. Then discard the funnel.
13. Place 1 drop of reagent 1 in the center of the insert
board well, on top of the cell button. Close the cyto-
block cassette and place it in fixative to await tissue
processing.
14. After processing in the standard tissue processor,
open the cytoblock cassette. Fold back paper and
remove the board insert. Use fine forceps inserted
through holes under the board insert.
15. Dislodge the cell button into the base mold and
embed flat. Discard the board insert and backing paper.
16. Reclose the cytoblock cassette and place
flat side up
(round peg side down) on top of base mold. Fill with
paraffin.
17. Handle as any paraffin block, but trim carefully
because cell buttons may be quite thin.
Preparation of cytospins for Tumor Markers60
1. Aspirates for cytospin preparation and tumor markers
are received as a needle rinse into balanced salt solu-
tion. Enough sample should be present so that the
fluid is slightly cloudy. If the sample is grossly bloody,
it should be saponized (see later). If the sample is
grossly cloudy, it should be diluted with balanced salt
solution until slightly cloudy before being used for
cytospin preparations.
2. Follow the manufacturer's directions to set up the
Shandon Cytospin.
3. Samples that are blood-tinged or have required
saponizing should be washed in balanced salt solu-
tion and centrifuged for 15 minutes at 1500 rpm.
Restore the original volume.
4. Break the cytospin filter seal by scraping your
fingernail around the edge of the circle on the filter a
couple of times. This allows for better absorption of
fluid.
5. Load the cytospin with sample chamber, filter, and
slides so the chamber is balanced.
6. Using a disposable pipette to dispense drops, add
specimen to the specimen chamber, usually 2-5
drops depending on cellularity of the sample.
7. Cellularity can be checked initially by using 1 drop
of sample on a slide and 1 drop of supravital stain.
Mix thoroughly with an applicator stick and cover-
slip. Examine under the microscope, and if the field
has 5-10 cells, then 1 drop of sample will provide
an adequate cytospin sample. If the cell estimate is
higher than this, dilute with balanced salt solution to
at least this distribution of cells or lower.
8. Follow addition of sample drops to cytospin chamber
with 2 drops of 20% fetal calf serum in RPMI (tissue
culture transport media).
9. Spin for approximately 5 minutes at speed of
500 rpm.
10. When cytospin stops, remove slides and filters.
Discard filters.
11. Allow slides to air-dry.
12. Determine again cytospin quality by using a repre-
sentative slide and staining it with Diff-Quik. Exam-
ine for adequate cellularity under the microscope.
13.If necessary, increase or decrease the number of sam-
ple drops used. Cells should not overlap, because this
provides confusing staining patterns and edge effects.
14. Prepare an adequate number of quality slides to proc-
ess the immunohistochemistry tests ordered.
15. Wash sample chambers in bleach, rinse with water,
and allow to dry after use.
Saponization
This procedure may be applied to visibly bloody fluids and may be
used in small amounts on samples that exhibit traces of blood.
593
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