asktheexpert
biopsy procedure and
sample preparation
BY SONIA CARBONE, B.SC, CT(ASC), CMIAC, SENIOR HOSPITAL SCIENTIST, & ANDREW FIELD, FRCPA,
FIAC, DIP CYT (RCPA), DEPUTY DIRECTOR & SENIOR STAFF SPECIALIST, DEPARTMENT OF ANATOMICAL
PATHOLOGY, ST. VINCENT’S HOSPITAL, NSW.
Dear expert, We are performing quite a number of fine needle biopsies in our workplace but there is much debate on the correct
procedure and methods of slide preparation. Can you help?
At some point during a career in ultrasound
every sonographer will come in contact with
ultrasound guided biopsies. Fine needle
biopsy (FNB) and core biopsy (CB) are the
two types of biopsies often performed when
sampling a region of interest.
It is vital that correct procedures are
followed and slide preparation is optimal
so that the maximum information can be
retrieved from sampled tissues. It is
the aim of this article to detail these
procedures.
Fine needle biopsy
FNB is a simple procedure that involves
passing a thin needle through the skin
to sample fluid or tissue from a cystic or
solid mass [1]. The sample of cellular
material is then deposited and smeared
on glass slides then fixed by air-drying or
immediate placement in alcohol. It is then
sent to a pathology laboratory for staining
with clinical, radiological and other
laboratory data. This pre-operative workup
may also include core biopsy. However,
FNB in some situations can substitute
for conventional surgical histopathology,
particularly when ancillary studies on FNB
include FNB of lung, pancreatic and
Equipment
thyroid carcinomas, metastatic lesions
Figure 1 demonstrates the set-up
required prior to beginning the procedure.
The equipment required includes:
in lymph nodes, high-grade lymphomas
with flow cytometry and cytogenetics,
carcinoma of the breast, when there is
correlation with imaging findings, and
• Frosted ended slides.
patients with recurrent tumours or who are
• Empty slide containers for air-dried
slides.
inoperable.
The diagnostic sensitivity and specificity
of FNB depends on several factors
including:
• The site and type of lesion.
• The experience of the operator
performing the biopsy.
• The quality of the specimen preparation.
subsequent analysis under a microscope.
• The diagnostic skills of the
cytopathologist.
a sample and produce on the microscopic
slide stained material that is a reliable
reflection of the disease process.
Today, FNB is regarded as a minimally
invasive, cost-effective technique with
diagnostic accuracy in the range of 90%
to 99% [2]. FNB is usually a component of
the pre-operative and pre-treatment study
of pathological processes in combination
14 issue 3, 2007
Fine needle biopsy technique
material are available. Such situations
using Giemsa or Papanicolaou stains and
The fundamental requirement is to obtain
cases, the cutting action of the needle and
the inherent pressure within the tissue
of the lesion will produce material in the
needle hub without any aspiration.
The commonly used term fine needle
aspiration biopsy (FNAB) is in some
ways a misnomer, because it is the
cutting action of the needle, moved many
times rapidly into and out of the lesion,
that produces material in the needle to
be deposited on the slide, rather than
‘aspiration’, which tends to enhance blood
contamination of the specimen. In many
• Slide containers containing 95%
alcohol or modified Carnoy’s fixative.
• RPMI transport medium/Hanks salt
solution in a container.
• Request form.
• 23/25 gauge (G) needles.
• 10ml syringes.
Fig 1. The set up of equipment prior to FNB.
soundeffects
biopsy procedure and sample preparation
Modified Carnoy’s fixative contains 5%
acetic acid, which lyses red blood cells,
needle, local anaesthetic is not usually
required.
which can obscure cellular detail in alcohol
fixed slides. The slides can remain in this
The procedure
fixative for up to 48 hours, but for longer
All ultrasound gel should be wiped away
from the area as this will be aspirated into
the needle, deposited onto the slide and
will obscure cellular material rendering
the sample uninterpretable (figs 2 and
3). The skin should be wiped with an
antiseptic, such as an alcohol swab.
transport 95% alcohol is preferred. Larger
gauge needles, such as 18, 20 or 22,
should NOT be used. Using a larger gauge
does not increase yield in bloody or hard
sclerotic lesions where a 23, 25 or even
26 gauge needle is recommended. Spray
fixative is not recommended for alcohol
fixation.
Fig 4. Cameco syringe pistol, 25G needle and
syringe. Adapted from [3].
Preliminary preparation
• With a lead pencil, label the frosted
end of the slides to be used with the
patient's name, date, date of birth and
site of biopsy.
• Label the container of RPMI transport
medium with the same details.
• If more than one site is to be biopsied,
label slides and containers specimen
1, 2, etc., and with the appropriate site
of the biopsy.
Fig 2. Microscopic material obscured by
ultrasound gel.
• Read the request form to see what
tests the doctor has requested.
These may include microbiology, flow
cytometry, cytogenetics or cell block.
If these ancillary tests are requested it
is best practice to dedicate a separate,
additional pass to each test.
Fig 5a. Sampling with aspiration. A needle is attached
to a syringe. The plunger is retracted by hand to
create negative pressure to allow aspiration of
material into the hub of the needle. Adapted from [4].
The puncture
Good quality fine needle biopsies are
obtained when the cellular material
obtained is retained within the needle.
It is important to avoid aspirating the
material into the syringe and, if possible,
avoid diluting the aspirate with blood
or fluid. If the lesion is cystic, the cyst
should be drained using aspiration, and
any residual lesion re-biopsied.
At the first appearance of any sample in
the hub of the needle (at the junction of
the syringe and needle) the biopsy is
stopped and if aspiration is being utilised,
the syringe plunger is released allowing
the pressure in the syringe to equalise
with the lesion pressure.
It is important to preserve the quality of
the material obtained by making the
smears immediately after completing
the puncture. Because of the rapidity
of the procedure and thin calibre of the
soundeffects
Fig 3. Microscopic visible material of a small cell
carcinoma.
Step 1. Insert the needle through the skin
into the centre of the lesion. The operator
with experience will feel the difference
between the firmness of the tissue and
the lesion being sampled; for example,
the needle passing through the capsule
of a lymph node, into the soft lymphoid
tissue, or the firm ‘gripping’ texture of a
fibroadenoma. The syringe can be held
in a syringe holder or ‘gun’ which allows
negative pressure to be obtained within
the syringe (fig 4), or the needle can be
attached to a syringe, either directly or with
plastic tubing, and held by the operator or
an assistant. If using a needle attached to
the syringe within a syringe holder, ensure
the syringe plunger is fully compressed
before starting the procedure.
Fig 5b. The plunger is retracted with the use of a
Cameco syringe pistol. Adapted from [4].
issue 3, 2007 15
biopsy procedure and sample preparation
(a)
(b)
(e)
(c)
(d)
(f)
(g)
Fig 6. Summary of fine needle biopsy proce�
forth in the lesion, directing it at different ar�
is then r�
Step 2. When the needle is passed
into the lesion, it is rapidly agitated
forwards and backwards in the one
plane. This rapid movement may produce
50 to 100 passages into the lesion in
less than 60 seconds. If there is no
material in the hub of the needle, the
plunger of the syringe is then retracted,
creating negative pressure for aspiration
(figs 5a & b). If fluid appears in the hub
of the needle immediately the needle
is placed into the lesion, the cystic
component can be drained.
Step 3. To sample the lesion more fully,
the angle of the needle can be altered, but
only when the needle has been withdrawn
to the edge of the lesion. It can then be
directed into a different region of the
lesion. Negative pressure can be applied
in the syringe as appropriate by keeping
the plunger retracted.
This phase is completed when material, fluid
or blood appears in the hub of the needle.
Step 4. When material appears in the hub
of the needle, the pressure in the syringe is
allowed to equalise by gently releasing the
plunger of the syringe, before the needle is
withdrawn from the lesion and skin.
Never withdraw the needle from the lesion
when any negative pressure remains in
16 issue 3, 2007
dapted from [3].
the syringe, because the sample in the
needle will be sucked into the barrel of the
syringe, where it cannot be retrieved onto
the slides. If material is sucked into the
syringe, this can be recovered by rinsing
with RPMI medium, and then cell block
preparation.
A summary of the needle biopsy
procedure is shown in figure 6.
Slide preparation
Fig 7a.
Smears should be prepared immediately
after the needle has been withdrawn
from the skin, to prevent clotting of the
specimen, air-drying and to ensure good
preservation of the cellular material
obtained (figs 7a & b).
After the needle has been withdrawn, the
syringe is disconnected from the needle,
filled with air and reconnected. To avoid
needle stick injuries, the hand should
commence on the syringe barrel, moving
the fingers down to the needle hub, which
is gripped and turned to release the needle.
The needle is held almost at right angles to
the slide and the material in the hub gently
expelled (fig 8), care being taken to deposit
it as a single drop 10mm from the frosted
label edge, approximately at the junction of
2/5th to 3/5th of the slide length.
Fig 7b.
Figs 7a & b. Compares air-dried (a) to well-preserved
microscopic cells (b) of apocrine cells seen in cystic
lesions of the breast.
soundeffects
biopsy procedure and sample preparation
Finally, the needle and syringe are rinsed
with RPMI transport medium by partially
filling the syringe with RPMI and expelling
the solution gently back into its container
(fig 11).
Fig 8. The needle is held close to a right angle to the
slide and material is deposited in a single drop.
Gently lower another slide onto the bottom
slide where the specimen has been
deposited, then quickly but gently pull
top and bottom slides apart as the drop
spreads from the weight of the top slide
(fig 9).
Fig 9. Shows top slide being placed on the bottom
slide. The material is gently spread from the weight
of the top slide.
Smears can then be:
• Immediately immersed in alcohol
fixative. The split second the smeared
slides are separated, the bottom slide
should be deposited in the alcohol
fixative (fig 10) or;
• Allowed to dry using a gentle warm air
stream from a simple hair-dryer.
Fig 12a. Shows a section from a cell block from a
FNB lymph node stained with haematoxylin and eosin.
Fig 11. Shows the needle and syringe being
rinsed in RPMI medium.
Further needling (a second or third
pass) may now be performed using a
new needle and the slide preparation
procedure repeated.
All specimen containers should have
the lids screwed on correctly and the
slides/specimen containers should be
unambiguously labelled with the patient’s
name, date, site of biopsy and the number
of passes. Complete a request form
including patient’s name, address, date of
birth, exact site of biopsy, tests requested,
referring doctor plus any relevant clinical
information. The specimen should be sent
immediately to the cytology laboratory for
processing.
Additional material collected at FNB
Any additional material collected can be
prepared in the following ways.
Needle rinse
Cellular material rinsed out of the syringe
in the RPMI solution can be centrifuged,
and used to make either cytospin slides or
a cellblock, depending on the size of the
pellet after centrifugation.
Cytospin slides are stained using various
stains to aid diagnosis, while a cell
block is fixed in formalin, embedded in
paraffin, cut into sections and stained
with haematoxylin and eosin or with
immunostaining (fig 12a).
Fig 10. Shows one slide being placed immediately in
alcohol fixative, while the other is allowed to air-dry.
soundeffects
Immunostaining is an extremely important
ancillary tool for the definitive diagnosis of
lesions. For example, carcinomas can be
stained to elucidate the exact site of origin
Fig 12b. Shows a section from the same cell block
showing positive staining CAM 5.2 – keratin for
metastatic carcinoma.
of metastases in lymph nodes (fig 12b).
Microbiology
If clinically it is suspected that the lesion
could be due to an infection a sample
should be taken for microbiological
analysis:
• If purulent fluid is present, then place
any excess fluid in a sterile yellow top
container.
• If there is little fluid obtained then place
10ml of sterile saline in a sterile yellow
top container and then rinse the needle
and syringe in this fluid in the same way
as you would for a needle rinse.
Flow cytometry
If clinically you suspect a lymphoma, as
in a FNB of a lymph node or para-aortic
mass, then a sample for flow cytometry
should be collected.
This sample is best collected in RPMI
medium in the same way as detailed
above where the cellular material in
the needle and syringe is rinsed in the
solution and placed in a correctly labelled
specimen container. Cytogenetics can also
be performed on the sample.
issue 3, 2007 17
biopsy procedure and sample preparation
Note: RPMI medium or Hanks salt solution
should be kept in the refrigerator and will
turn a yellow colour when it has expired.
Points to remember
1. Release the negative pressure in the
syringe before withdrawing the needle
from a lesion. Otherwise the collected
cells will be pulled into the syringe
causing air-drying artifacts and making
it impossible to retrieve the material
from the syringe for cytology.
2. Disconnect the needle from the syringe
to retract the plunger, filling the syringe
with air, reconnecting the needle and
expelling the material onto the slides.
Material will be lost in the syringe if this
is not done, causing the same problem
as in (1).
3. Do not allow the material to clot in the
needle once it is withdrawn from the
patient. Immediately expel the material
onto the slides. The operator should not
keep the needle in the patient for more
than 60 seconds because the material
will clot in the needle and, with each pass,
this time limit should be even shorter.
4. Do not allow the material to air-dry prior
to, during, or after making smears,
unless purposely preparing air-dried
slides. There should be minimal time
delay between completion of the biopsy
and preparation of the slides. Smears
for alcohol fixation and Papanicolaou
stained slides should be immediately
placed in alcohol fixative. All air-dried
slides should be completely dry before
they are placed in a container and
transported to the laboratory.
5. Slides and RPMI medium are not
sterile; hence a new needle should be
used for each pass, and each different
site biopsied.
6. If cyst fluid is obtained in the syringe,
the needle need not be disconnected to
retract the plunger prior to making the
smears. Smears may be made directly
with a few drops of the fluid expelled
gently onto the slide to form a droplet
approximately 8mm in diameter. The
remaining fluid should be sent to the
laboratory in a labelled container for
cytospin slide preparation and routine
processing.
7. Do not rinse or lubricate the needle
and syringe with any substance prior
to biopsy. This may cause destruction
of part or all the cellular material by
osmotic cytolysis.
8. Send the specimen with a signed
request form detailing the relevant
patient information and clinical history.
It is important to remember that the
effectiveness of FNB is largely operator
dependent and it is important to have all
equipment ready to minimise any potential
problems with specimen preparation.
Because an FNB can only sample a region
of a mass or lump, there is a risk that any
abnormal cells may be missed and not
detected through sampling error. Making
three or four separate passes into a lesion
can reduce this error.
[2]. It is sometimes used instead of
FNB, or visa versa, but the tests are
complementary. FNB is less expensive
and offers immediate staining and
assessment. This creates the opportunity
for triaging, extra FNB passes or core
biopsy. FNB is generally less traumatic
to the patient with less bruising.
Core biopsy is a more invasive procedure
than an FNB, as it involves making a small
incision in the skin after local anaesthetic
and a wider bore cutting needle.
The samples of tissue taken during a
core biopsy are collected into a specimen
container filled with 10% buffered formalin
and sent to the histology laboratory for
processing. This processing generally
takes 24 to 48 hours.
Conclusion
These guidelines have been developed
from experience at St. Vincent’s Hospital
and we hope you find them useful. It is
vital to consult your current pathology
service and it is important that you follow
the guidelines of your provider.
References
1. Virtual Medical Centre [homepage
on the internet]. Summary of Fine
Needle Aspiration Biopsy (FNA).
[updated 2007 Feb 20; cited 2007
Jul 1]. Available from: http://www.
virtualcancercentre.com/investigations.
asp?sid=3.
2. University of Iowa Hospitals and
Clinics. Fine Needle Aspiration Biopsy.
[serial on the internet]. 2002 [cited
2007 Jul 1];3(2):[about 4 p.]. Available
from: http://www.uihealthcare.
com/news/currents/vol3issue2/
05fineneedleaspiration.html.
3. Orell SR, Sterrett GF, Walters MN,
Whittaker D. Manual and Atlas of
Fine Needle Aspiration Cytology. 2nd
ed. Edinburgh: Churchill Livingstone;
Core biopsy
Core biopsy is another method of tissue
diagnosis; that is, a way of sampling
the cells in a suspicious lump or mass
18 issue 3, 2007
1992.
4. Trott PA. Breast Cytopathology. A
Diagnostic Atlas. London: Chapman &
Hall Medical; 1996.
soundeffects