20
Fine-Needle Aspiration Biopsy Techniques
15.3% sodium hydrogen peroxide
16. Cytospin slides (Shandon)
17. Cytospin 3 (centrifuge for making cytospins
Shandon)
W orking Solutions
1. Dako target retrieval solution (1x)
a. 100 mL of Dako concentrated target retrieval solu-
tion (10x)
b. 1000 mL of distilled water
Prepare fresh everyday
2. Dako target retrieval solution pH 9 (1x)
a. 100 mL of Dako concentrated target retrieval solu-
tion (10x)
b. 1000 mL of distilled water
Prepare fresh everyday
3. Wash buffer solution
a. 100 mL of Dako wash buffer concentration (10x)
b. 1000 mL of distilled water
Expiration: 5 days
4. 5% normal goat serum
a. 5 mL of normal goat serum (Dako)
b. 95 mL of reconstituted Sigma buffer
c. 2 drops of blue food coloring
Expiration: 1 month
5.
DAB chromogen solution (Dako K3466)
a. 1 drop of DAB chromogen
b. 1ML of substrate buffer
Make fresh every day.
Controls
Using one unstained slide from each case, the primary antibody
is replaced by a cocktail negative control antibody containing
anti-mouse and anti-rabbit immunoglobulins. All other immu-
nohistochemical-staining steps are carried out according to this
protocol. The "negative control" slide serves as a test for non-
specific binding of immunoglobulins. Because most immuno-
histochemical stains are run in panels that include several
primary antibodies applied to several slides, the slides stained
for antibodies that do not react with the cells in question serve as
additional negative controls for the immunohistochemistry pro-
cedure. For each primary antibody, histologic sections of various
tumors and normal tissue known to contain cells positive for
the antigen detected by the primary antibody are stained for that
antigen. These positive control tissues serve as tests of the specifi-
city and immunoreactivity of the primary antibodies.
The tissue sections, when possible, are placed on the same
slide as the patient tissue sample. In addition, patient sample
sections stained for various antigens often contain normal tissue
References
1.
Frable WJ. The history of fine needle
aspiration biopsy: The American experience.
In: Schmidt W (ed.)
Cytopathology Annual
1994.
Chicago: AsCp Press; 1994.
2.
Gupta S, Henningsen JA, Wallace MJ,
et al. Percutaneous biopsy of head and
neck lesions with CT guidance: various
structures immunoreactive for the antigen detected by each
antibody used in the immunohistochemical stains selected to
evaluate the individual case. The immunoreactivity of the pri-
mary antibody and of the entire immunohistochemical staining
protocol is applied to a specific case.
procedure
la. Cut paraffin sections at 3 pm and mount on charged
slides that have a control tissue section at the top in
the "control" section of the slide. Label slide with case
number, block number, and stain.
lb. Prepare Cytospins (see previous description for
cytospin preparation).
2a. Dry paraffin slides in 60°C oven for minimum of 60
minutes.
2b. Cytospins, aspirates, and smears are fixed in anhy-
drous acetone for 15 minutes at 4°C then air-dried
for a minimum of 35 minutes.
3. Deparaffinize sections in 3 changes of Xylene—five
minutes each.
4. Rehydrate section in decreasing grades of alcohol for
3 minutes each: 100% two changes, 95%, 80%, and
then rinse in distilled water.
5. Treat slides in 3% hydrogen peroxide for five minutes.
6. Paraffin section specimen pretreatment: Place slides
in heated target retrieval in steamer. For target
retrieval: 20 minutes; for pH 9: 30 minutes. Allow
slides to cool on counter for 20 minutes, and then
they may be placed on the autostainer.
7. Block nonspecific protein binding sites by incubation
for 5 minutes in normal goat serum.
8. Rinse for 2 minutes in Dako wash buffer.
9. To the test slide add appropriate primary antibody
using the optimal dilution in antibody diluent, and
to the negative control slide add the anti-mouse/anti-
rabbit immunoglobulins. Be sure that the section is
completely covered with solution for recommended
incubation times.
10. Rinse for 2 minutes in Dako wash buffer.
11. Add HRP Envision polymer, incubate for 20-50
minutes (incubation time varies, depending on the
primary antibody).
12. Rinse in 2 changes of Dako wash buffer 2 minutes
each.
13. Place slides in DAB or DAB+ substrate solution for
5 -8 minutes. Substrate should be prepared just
before use. (Incubation times and chromogens vary
depending on primary antibody.)
14. Rinse for 2 minutes in Dako wash buffer.
15. Counterstain slides in Gill's III hematoxylin for
30 seconds.
16. Blue slides in running tap water for 2-3 minutes.
17. Dehydrate, clear and cover slip.
approaches and relevant anatomic and
technical considerations.
Radiographics
2007;27:371-390.
3.
Vilmann P, Saftoiu A. Endoscopic
ultrasound-guided fine needle aspiration
biopsy: equipment and technique.
J Gastroenterol Hepatol
2006;21:1646-
1655.
4. Herth FJ. Mediastinal staging—the role
of endobronchial and endo-oesophageal
ultrasonic graphic guided needle
aspiration.
Lung Cancer
2004;45(suppl
2):S63-67.
595
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