Section C

Fine Needle Aspiration of CHAPTER 20

Various Organs and Body Sites

Fine-Needle Aspiration Biopsy Techniques

William J Frable

Contents
History of Aspiration Biopsy Concluding Remarks
Clinical Skills Required Appendix
The Thin-needle Aspiration Method Equipment
Training and Planning Staining Techniques
Basic Equipment 1. Papanicolaou Stain
Ancillary Equipment and Special Procedures 2. Rapid Papanicolaou Stain46
Aspiration Technique 3. Diff-Quik Stain Set
Performing the Aspiration 4. Modified May-Grünwald-Giemsa Stain
Smear Preparation 5. Hematoxylin-Eosin Stain

Fixatives and Stains Other Techniques

Cell Block Preparation
Ancillary Techniques and Applications
Preparation of Cytospins for Tumor Markers60
Organization of the Aspiration Biopsy Service Saponization
Complications of Fine Needle Aspiration Biopsy Supravital Stain
Immunostaining of Cytospins Using the Dako Autostainer

History of Aspiration Biopsy

The use of fine-needle aspiration (FNA), a method of aspira- Martin and Ellis in 1930 are generally credited with the first
tion biopsy cytology, continues to grow both in North America description of sampling of tumors by means of a narrow-gauge
and throughout the World.1 Improvements in imaging, com- needle.16 This method of tumor diagnosis became quite popu-
puted tomography scan (CT), and ultrasound (US) have fueled lar at Memorial Sloan Kettering Cancer Center through the late
the growth of FNA among both interventional radiologists and 1950s, but was not accepted in any significant way in other US
clinicians.2,3 Endoscopic ultrasound is already used widely by medical centers.17 The history of aspiration biopsy in Europe
gastroenterologists and radiologists to investigate lesions within extends back to the mid-nineteenth century, preceding the
both the abdomen and chest.4–7 An increasing number of ­targets invention of the microtome, which then allowed thin sections
for aspiration biopsy are discovered incidentally during investi- of tissue to be cut.18 Almost immediately after World War II, an
gation of the abdomen and chest for often nonspecific or vague experienced group of Swedish clinicians trained chiefly in hema-
symptoms. The term “incidentaloma” has gained credence in tology and oncology led a resurgence of the application of aspi-
the medical literature, primarily for incidental adrenal lesions, ration cytology.19-21 Aided by several clinicians and pathologists
though this term appears in no recognized classification of who studied with the group in Sweden in the early 1970s, FNA
either neoplastic or non-neoplastic diseases.8,9 The dominant again gained considerable prominence in the United States as
clinical sites for FNA still remain breast, thyroid, and lymph a useful biopsy method for tumors.22 Growth of imaging meth-
nodes among superficial tissues. The availability of high-resolu- ods and the specialty of interventional radiology has certainly
tion mammography with stereotactic needle placement has also provided additional fuel to the expansion of aspiration biopsy
focused renewed attention on aspirating non-palpable lesions and has in recent times shifted this biopsy method away from
of the breast, although the emphasis has shifted over the past exclusively aspiration and toward microcore biopsies either
few years to core needle biopsies.10–13 Large bore needles are also alone or in combination with aspiration.23-26 Some comprehen-
being used to both biopsy and excise small breast tumors, lead- sive technology assessment of aspiration biopsy has now been
ing to challenges to surgical pathologists to evaluate margins in carried out. FNA is generally cost-effective for superficial masses
fragmented specimens and assess the risk of invasion when it but may be only marginally so for deeper lesions where both
cannot be detected microscopically.14,15 pathologists and radiologists are involved and considerable

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time may be required to localize and obtain a suitable specimen review the data files of their laboratory for any prior accessions
for interpretation.27-30 While there is now an increased availabil- that may be pertinent to the present clinical situation.
ity of well-trained cytopathologists, their ability to establish a Cytotechnologists may be quite important to an aspiration
pathology-based aspiration service, at least within the United service, ensuring that smears are properly prepared and fixed and
States, has been limited by questions of efficiency of such a serv- that material for cell block, immunohistochemistry, or electron
ice, consolidation of pathology practices into large groups and microscopy as well as other specialized techniques is handled
laboratories of national scope, and the structure of current reim- appropriately. At a minimum this requires cytotechnologists to
bursement system for medical care. The dramatic increase in the be able to evaluate aspirates taken by other clinicians for cellu-
use of aspiration biopsy cytology can easily be determined with larity so as to triage the aspiration samples appropriately. Cyto-
just a casual review of journals devoted primarily to cytopa- technologists may also screen aspiration smears. While cellular
thology or the meetings of major pathology and cytopathology smears do not require this activity, those smears either of low
societies.1 Ready application of new technology, immunohisto- cellularity or diluted by blood and fluid benefit from system-
chemistry, and molecular diagnostics to aspiration cytology has atic screening by the cytotechnologist. Aspirates submitted in a
also helped to refine our ability to interpret small samples and liquid preservative for the preparation of monolayers likewise
provide very definitive diagnoses.31,32 benefit from screening as diagnostic cells may be limited and
patterns for interpretation are lost in these types of prepara-
tions.
Clinical Skills Required Despite the recognized participation and value of cytotech-
nologists to an aspiration biopsy service, it is the author’s firm
Aspiration biopsy may be indicated whenever there is a palpa- belief that the pathologist must be actively involved in the aspi-
ble tumor mass or a lesion visualized within any organ. For the ration biopsy, making both the initial and final evaluation of the
physician or more specifically for the pathologist performing smears.36 Good quality aspirates and correctly prepared smears
FNA, some familiarity with general anatomy is essential. For the usually result in more than adequate cellularity in a confined
pathologist performing this biopsy some sharpening of clinical area on the slide that does not require screening. The major-
skills, both obtaining a focused clinical history and performing ity of FNA interpretations may be based on pattern recognition
a physical examination are required. The cytopathologist must rather than the features of single cells and thus reflect a close
also have a good reference frame for normal cell elements from relationship to the interpretation of histologic sections used for
a variety of organs and tissues as they appear in smears. To inter- conventional surgical pathology.17
pret aspiration biopsy smears, the pathologist also needs a com-
prehensive knowledge of surgical pathology, thus making he or
she able to translate from traditional tissue patterns of lesions to The Thin-needle Aspiration Method
their appearance in aspiration smears.33
Some experience with smear patterns may be initially acquired Thin needles, 0.6–1.0 mm., generally 22, 23, 25, and 27 gauge,
by making touch imprints from surgically excised tumors and/or are used for the performance of aspiration biopsy, most often
acquiring aspiration samples from normal tissues or neoplasms 1.5 in. in length. Special situations may dictate shorter needles
submitted for surgical pathology examination. This “practice” and even higher gauge. For example, the very small cutaneous
is an important step in becoming skilled at taking, preparing, metastasis of breast carcinoma on the chest wall may be sam-
and staining aspiration biopsy smears.34 FNA may encom- pled more easily with a 27-gauge, 1-in. or even ½-in. needle and
pass practically all tumor types, both benign and malignant, with a small, 3.0- to 5.0-mL syringe, approaching the nodule
observed in the general practice of surgical pathology. Build- in a plane perpendicular to the skin surface, in the manner of
ing a reference file of smear preparations from the specimens performing a tuberculin skin test. The vitreous may be aspirated
found at the surgical pathology cutting bench is therefore quite by ophthalmologists using a 30-gauge needle to obtain a very
useful when confronted by actual clinical cases. This surgical small sample that is useful for diagnosing metastatic or primary
­pathology–cytopathology interface cannot be overemphasized neoplasms within the eye.37
if the pathologist expects to become successful with aspiration With the continued growth of fine-needle aspiration biopsy
biopsy cytology.35 Clinicians performing aspiration biopsy obvi- there have been some variations in needle design. The Franseen
ously lack this essential ingredient of experience and knowledge needle has a notched tip and stylus, which in practice seems to
of morphology. There is no other subspecialty where such a provide both semisolid material for smears and sometimes pre-
­combination of clinical skills and morphologic ­ interpretation dominantly thin microcores of tissue. These microcores, what-
interface for a high level of success. ever style of needle provides them, should be rolled onto the
The surgical pathologist or cytopathologist initiating an aspi- slide surface to obtain smears for initial staining and examina-
ration biopsy service will have to relearn history-taking skills. tion. They should not be crushed between two slides in an all-
While the focus is on the story of the lump or bump, the patient’s out effort to produce smears.
complaint, other background information may be of impor- The Milex and INRAD needles were designed to improve
tance. As an example, a child or adult with an enlarged lymph sampling from firm fibrous breast masses. They both have a slot
node may have had exposure to a cat. If the aspirate shows a on the side and are referred to as side-port needles. While the
reactive pattern of lymphoid cells and/or has granulomas, this side-slotted needles procure a greater volume of aspirate they
is quite compatible with cat scratch disease. Obviously for any also produce much more discomfort during aspiration. The
patient with a newly discovered mass the pathologist needs to author does not use them for that reason. Use of a small amount
inquire about any previous history of a neoplasm or symptoms of local anesthetic may be indicated when aspirating with the
that may point to a particular organ. The pathologist must also side-port needles.

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Fine-Needle Aspiration Biopsy Techniques

Radiologists most often use the Chiba needle of 21 and 22 should welcome the opportunity to see patients for that is the
gauge for transthoracic and transabdominal aspirations. Within best way to obtain a complete clinical picture and then correlate
the past several years, and as imaging has become more precise, it with the biopsy material to arrive at a correct interpretation.
radiologists have gotten bolder, employing the Franseen needle The same approach is equally important for interpretation of
of either 20 or 21 gauge or even a more standard core needle tissue biopsies in surgical pathology.
of 18 gauge. Fine-needle aspiration biopsy of the prostate has
disappeared in the United States, giving way to transrectal ultra-
sound-guided core needle biopsies using a 21-gauge needle.38
Basic Equipment
Microcore needle biopsy for both palpable and non-palpable The basic equipment used for rapid and efficient performance of
breast lesions has largely supplanted fine-needle ­ aspiration thin-needle aspiration biopsy are as follows.
biopsy, though controversy over which method remains.39,40
1. Cameco Syringe Pistol, Aspir-Gun, or other type
In medical centers that specialize in diagnosis and treat-
­aspiration handle (see Appendix);
ment of bone and soft tissue tumors, core needle biopsy is often
2. Ten- or 20-mL disposable plastic syringe with ­LuerLok
combined with FNA.41–44 If the cortex of the bone is intact, it is
or straight tip, depending on aspiration gun handle
necessary to drill a hole through the cortex before aspiration
size;
or the very-large-bore bone marrow-type core biopsy needle is
3. Twenty-two- to 27-gauge, 0.6- to 1.0-mm external
used. Local anesthesia must be administered before biopsy of
diameter disposable needles, 3.8 and 8.8 cm, 15 and
a bone by either fine-needle aspiration or a core biopsy, and
20 cm long, with or without stylus; the needle hub
local anesthesia is also useful before attempting FNA of soft tis-
should be clear;
sue tumors, many of which are both large and deeply situated
4. Alcohol skin preparation sponges; betadine skin
within the proximal muscles or other soft tissues of the trunk.
sponges for deeper aspirations, transabdominal, tran-
As a general rule, the larger the external diameter of the
sthoracic, bone (where the cortex is not intact or the
­needle, the greater the likelihood of complications with needle
periosteum is elevated), or deep soft tissue;
biopsy. Increasing the radius of the needle likewise increases
5. Sterile gauze pads;
the cross-sectional diameter exponentially. If one employs only
6. Microscopic glass slides with frosted ends;
the thin-needle technique, there are virtually no complications,
7. Small vial of balanced salt solution and/or RPMI
the exceptions being FNA of the thorax (pneumothorax) or
­tissue culture transport media;
some cases of excessive bleeding with transabdominal aspira-
8. Suitable alcohol spray fixatives for immediate fixation
tion biopsy.45
of wet smears (note that immediate fixation is not
required when using the Yang rapid Papanicolaou
Training and Planning stain);46
9. Ten- or 20-mL capped tube with 10% neutral ­buffered
Pathologists planning to practice aspiration biopsy should have
formalin for cell-block or microcore sample fixation;
a good knowledge of anatomy. They should retrain themselves
this is not absolutely necessary as material placed in
in history-taking and physical examination. The majority of
RPMI can be converted to a cell block in preference to
palpable masses amenable to FNA are located in a superficial
the preparation of cytospins as dictated by the initial
position, or they are in an area not in direct relationship to
evaluation of smears;
normal structures, major arteries for example, that would make
10. Optional vial of local anesthesia, 1-2% lidocaine;
the biopsy procedure potentially hazardous. The site of punc-
topical spray anesthesia for aspirates in children or
ture of the aspirating needle should be planned in a manner
intraoral aspirates; vials of lidocaine that dentists use
that takes into account possible future treatment options. When
for local anesthesia and the dispensing equipment
aspirating suspected primary malignant tumors, the needle track
may be useful; see Abele and Miller;47
should be placed in a location likely to be included in any sub-
11. Small vial of buffered glutaraldehyde for fixing
sequent excision. While there is very little documented evidence
­aspirate for electron microscopy if required or
of needle track seeding following aspiration by the thin-needle
­anticipated.
method, selecting the proper approach to the target of a sus-
pected malignant tumor is still important to avoid that potential A small plastic tray easily holds all the equipment. Local
­complication. anesthesia is required for needle aspiration of transthoracic or
Any patient presenting for needle aspiration biopsy should transabdominal masses but is rarely necessary for other clini-
have their clinical problem thoroughly reviewed by the aspira- cally palpable lumps, though radiologists and clinicians using
tor. The pathologist trained to perform needle aspiration biopsy ultrasound to guide aspiration biopsy of the thyroid seem to
should take ample time in reviewing the history of the lesion prefer the use of local anesthesia. The author considers the use
to be biopsied. Careful consideration of clinical information of both ultrasound and local anesthesia when aspirating pal-
from the patient often provides clues as to whether the mass is a pable masses at any location a totally unnecessary step. How-
benign or a malignant neoplasm, is metastatic from a previously ever, those pathologists who have set up free-standing aspiration
treated malignancy, or is unlikely to be a neoplastic process at biopsy clinics prefer to inject local anesthesia in all patients.
all. This is the initial step in the process of arriving at a differen- They employ the equipment used by dentists and available from
tial diagnosis and should not be neglected. dental supply houses, 30-gauge disposable needles, 2-mL dis-
Surgical pathologists–cytopathologists with particular inter- posable cylinders of 2% lidocaine hydrochloride, with or with-
est in the study of tumors will find the application of needle out epinephrine, and a reusable metallic injection handle. Local
aspiration biopsy both a useful and rewarding contribution anesthesia can be very precisely dispensed in the area along the
to the management of patients with neoplasms. Pathologists planned needle track without much tissue distortion that might

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affect palpation of the aspiration biopsy target.47 For the most when the specimen is obtained. In this way, the biopsy may be
part, the method described avoids anesthetic burn. Also availa- ­properly handled, smears prepared and fixed appropriately with
ble are anesthetic canisters that dispense a short burst to deaden some smears stained for a preliminary interpretation. A mobile
only the skin surface. Use of this surface anesthesia may be help- cart with the appropriate equipment, supplies, and a microscope
ful when performing aspiration biopsy of masses encountered can be maintained in the pathology department for use in the
in children. various imaging suites in radiology or outpatient clinic areas, or
The author’s personal experience finds that aspiration biopsy taken to the operating room for intraoperative aspiration biopsy.
performed on superficial “lumps and bumps” without anesthe- While the ideal presented above maximizes the effectiveness of
sia is virtually non-traumatic. It is the unusual patient where the FNA, the reality today, with the growth of this procedure, is that
procedure may not be repeated frequently enough, usually three pathologists cannot efficiently cover all of these options, even
or four times, to obtain adequate amounts of material for both in large medical centers and group practices. Properly trained
diagnostic purposes and special tests (e.g. flow cytometry, immu- cytotechnologists or even pathology assistants can fulfill these
nohistochemistry). Some patients are apprehensive or have a obligations. However, it must be remembered that they can only
general fear of needles, have experienced syncopal episodes dur- provide adequate assessment and are not permitted to make
ing vena puncture, and therefore may require administration of definitive diagnostic interpretations, as that is the practice of
local anesthesia. A careful history taken before the aspiration medicine as defined by state regulations and licensure.
is performed, with the above items in mind, is important, as In the previously described situations it is the author’s view
is taking time to describe the procedure in detail and gaining that evaluation for adequacy is insufficient. The presence of the
the patient’s confidence. However, it has not been the author’s cytopathologist allows for an informed discussion of the clini-
experience that the use of local anesthesia ameliorates patient cal problem in terms of the aspiration sample obtained and the
apprehension. A careful explanation of the procedure is more resultant preliminary microscopic interpretation. For example,
likely to waylay the fear of needles or any medical procedure that it is of limited or of no value to pursue with multiple aspirates
may be encountered in some patients. An explanatory brochure a lesion that has clinical and radiographic features of a benign
provided to the patient before the appointment can be useful.47 process, when the evaluation of the material initially obtained
In no circumstances should an uncooperative patient undergo supports that interpretation. It is clear that cytotechnologists can
aspiration biopsy. This rule may be superseded in children, with be trained to handle the sample properly; they lack training and
parents’ permission, if the child can be restrained adequately. experience to correlate the clinical features of the case and inte-
The aspiration biopsy is usually described as similar to a vena grate those findings with the initial evaluation of the smears. This
puncture, for which anesthesia is not given and which is also is particularly true for intraoperative aspirations, the majority of
performed with larger, usually 16-gauge needles. Emphasis that which are for intra-abdominal masses, most often either within
the needles for aspiration biopsy are much smaller is important the pancreas or associated with the biliary tree. Radiologists (and
in the description and explanation of the procedure. surgeons in the case of intraoperative aspirations) should not
There are some variations in the use of fixatives for smears. prepare smears. They are not trained to make smears properly
The author prefers that the majority of the smears to be air-dried and rarely are amenable to spending the time to learn the correct
and later stained with a Romanowsky method, the Diff-Quik techniques. The common alternative today of placing the entire,
stain being preferred. Some smears are usually wet-fixed in 95% usually bloody, sample into some sort of fixative, frequently one
ethyl alcohol or sprayed using a commercial fixative contain- for liquid-based cervical cytology samples, sending it off to the
ing alcohol and 2% polyethylene glycol, the liquid form of laboratory with often minimal, illegible clinical information
Carbowax. With either of these fixation methods, cells are lost scribbled on a gynecologic or general cytology form, is utterly
both in the fixative solution or subsequently during staining. unsatisfactory. From the experience of the author’s consultation
An alternative, particularly for clinicians or radiologists, is for practice, the overwhelming majority of the problems with FNAs
all smears to be air-dried. Some of these smears are then stained are poor quality preparations with inadequate clinical infor-
with a rapid Papanicolaou method after brief rehydrating of mation. More often than not, the pathologist resides only in
them in normal saline as the first step in the staining proce- the laboratory and accepts whatever type of sample is sent for
dure. This method, published some years ago by Yang, produces evaluation. This is not in the best interest of patient care and
good nuclear detail comparable to that found in smears wet- significantly reduces the effectiveness of aspiration biopsy. The
fixed directly in alcohol, or spray-fixed. This rapid Papanicolaou astonishing growth of ultrasound-guided aspiration biopsy of
stain has an additional advantage of lysing red blood cells which the thyroid and other palpable masses by both radiologists and
effectively reduces the bloody background of some smears that clinicians would top the list of misused technology.
can make them difficult to interpret.46 A variety of techniques that have evolved through cell
research have applications to aspiration biopsy samples. They
may enhance the specifics of diagnosis and prognosis, particu-
Ancillary Equipment and Special Procedures larly in the case of malignant neoplasms.48–53 For most tumors,
The increasing use of imaging techniques both within the hos- aspiration samples are of adequate cellularity for flow cytom-
pital and in outpatient clinics has made it essential that there etry that today can determine cell lineage, most notably in the
be close cooperation between the cytopathologist and the inter- interpretation of lymphoproliferative disease. Over time the
ventional radiologist, or other clinicians who employ ultra- use of flow cytometry to determine ploidy and S-phase fraction
sound to locate and determine the nature of masses, particularly of tumors has proven less useful. Aspirates from breast cancer
endoscopic ultrasound. The author believes the cytopatholo- may be used to measure estrogen and progesterone receptors
gist should be present at the following procedures: ultrasound- as well as Her-2neu, using modern image analysis systems. The
and CT scan-directed deep aspirations and stereotactic-guided results are also quite comparable to those performed on the
sampling of non-palpable breast masses, at least at the time excised tumor.55,56 However, the accurate detection of Her-2neu

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Fine-Needle Aspiration Biopsy Techniques

has become so important in the management of breast cancer FISH and chromosome-specific probes.31,69,75 This method has
that great effort has been put into standardizing the method- also been used to quantitate the neu oncogene in transformed
ology to obtain meaningful and accurate results. FNA is not breast epithelial cells.76 For molecular diagnostic methods to be
included as a specimen type in recently published guidelines for effectively applied to aspirates or other cytologic samples, a ref-
testing Her-2 neu.57 erence laboratory or established medical center laboratory with
There continues to be remarkable growth in the number of an expert and interested staff needs to be available. Molecular
antibodies available for detecting cell products by immuno- diagnosis requires no special fixation or handling other than
histochemistry. Many of these reagents are equally applicable those routinely employed for preserving cytologic or tissue sam-
to aspiration samples, particularly cell blocks and/or micro- ples. Material from paraffin-embedded cell blocks can also be
cores.31,58 Immunocytochemistry can be performed on air-dried, used for FISH and a variety of other molecular methods. Cyto­
alcohol-fixed, or previously Papanicolaou-stained smears in spin preparations with adequate cellularity also work quite
an effort to successfully detect a number of antigens.59 While effectively for FISH.51,54,77–79
immunocytochemistry can be performed on direct smears,
more consistent results have been obtained in the author’s labo-
ratory with cytospin preparations, cell blocks, or microcores.60–62 Aspiration Technique
Low cellularity is the only limitation and is most often a factor
with cell-block specimens. It is therefore important to have the To be successful with an aspiration biopsy, it is important to
cytopathologists evaluate initial smears, determine at least a pre- ­follow the preliminary steps listed here:
sumptive diagnosis, and obtain additional samples as needed
1. Review the history of the patient. Determine the
to provide adequate cellularity for these special techniques. Par-
clinical problem and its relevance to the lesion to be
ticularly with lymphoproliferative diseases, adequate cells for
biopsied.
preparing a number of cytospins can be obtained by FNA and a
2. Determine whether the biopsy is justified.
panel of markers then used to characterize and aid in the diag-
3. Palpate the mass, attempting to determine its loca-
nosis of malignant lymphomas versus a reactive lymphoid pro-
tion in relation to surrounding structures. Estimate its
liferation versus another type of neoplasm. Good protocols for
depth. Decide on the optimal direction of the needle
the application of molecular methods for diagnosing Hodgkin’s
to accomplish the aspiration biopsy.
and non-Hodgkin’s lymphomas from aspiration samples have
been developed.63–66 A mass located deeply in tissue in usually best approached
While there have been advocates for ultrastructural study to perpendicularly to the skin surface. Small and superficially lying
characterize neoplasms from samples obtained by aspiration tumors are best approached by penetrating the skin at or very
biopsy,67 currently electron microscopy seems to have a very close to a horizontal plane, then feeling for the mass with the
limited role for the diagnosis of neoplasms both by aspiration needle tip.
biopsy and in the practice of general surgical pathology.68 The
4. The patient should be placed in a comfortable posi-
sampling technique for electron microscopy is, however, quite
tion for the aspiration biopsy, but the mass must be
simple. Simply obtain a separate aspiration sample and place
easily palpable and immobilized during the biopsy.
it directly into buffered glutaraldehyde fixative, centrifuge the
specimen, and handle the resulting cell button by standard tech- Step 4 is very important for head and neck lesions. The prom-
niques for electron microscopic studies. Because the aspirate inence of an enlarged lymph node, or lump, may sometimes
sample is directly and immediately fixed after being taken, the depend on whether the patient is supine or erect. The sternoclei-
quality of preservation of the cell organelles is excellent.52 domastoid muscle bulk and its close proximity to the cervical
Applications of molecular biology for the diagnosis of lymph nodes require positioning the patient such that the biopsy
­disease have continued to expand and increase both in surgi- needle passes through only a minimum of soft tissue and mus-
cal pathology and cytopathology.48,69 Detection of oncogene cle before reaching the target. Avoid aspirating a mass by travers-
c-erbB-2 in breast cancer and bcl-2 oncogene in lymphoid tissue ing the sternocleidomastoid muscle. It is painful and the needle
from FNA samples has been reported.28,55 Both fresh and archi- is likely to be plugged with fragments of muscle. The aspirate
val FNA samples (smears) have been successfully used.59,60,71 smears may look like a good sample was obtained but it is all
Determining clonality is quite useful in the diagnosis of lym- skeletal muscle. For the aspiration of thyroid lesions, it is usually
phomas, and these techniques have been applied to aspiration helpful to place a small pillow under the patient’s upper back,
biopsy samples.72–74 A portion or separate aspirate can be set extending the neck with the head tilted back. There is a groove
aside at the time the patient’s lesion is biopsied for the applica- formed between the lateral border of the trachea and the medial
tion of molecular diagnostics as needed. An expanding variety border of the sternocleidomastoid muscle. That is the area for
of probes for detecting gene rearrangements and specific tumor the aspiration of thyroid nodules within a plane perpendicular
markers are available. to the transverse vertebral process. The transverse vertebral proc-
One molecular method, fluorescent in situ hybridization ess provides a reference point for deep-lying thyroid nodules.
(FISH), is playing an expanded role in the diagnosis of neoplasms The aspirating needle may penetrate to touch bone of the trans-
from a variety of cytologic samples including aspiration biopsies. verse process, and then be withdrawn slightly. This should result
While much of the interest with FISH has been in finding added, in the needle being within the thyroid nodule. Penetration of
deleted, or translocated chromosomes as markers for urothelial the needle to the transverse process is not harmful or painful to
carcinoma detection, there are now a variety of relatively or very the patient. Remember to keep the needle in the perpendicular
specific chromosome abnormalities found in soft tissue and plane as described above. Do not direct the needle either medi-
other tumors that may be identifiable by FISH. Chromosomal ally, which may result in penetration of the trachea, or laterally,
abnormalities have been detected in tumors from FNAs using which could result in puncture of the carotid artery.

583
 PART TWO Diagnostic Cytology

5. Take time to examine the patient thoroughly. Discuss­ 3. Lay the syringe pistol with the appropriate gauge
your preliminary assessment of the patient’s lesion. needle attached to the syringe with the needle point
This is an opportunity to describe what will take against the skin and at the predetermined puncture
place during the aspiration and what is to be site and angle.
accomplished with it. A patient information bro- 4. Using a smooth but quick motion, insert the needle
chure describing the procedure is helpful and can be through the skin and into the immediate subcutane-
sent to the patient in advance of the appointment or ous tissue.
provided in the waiting room before the patient is 5. Next, advance the needle into the mass.
examined. It is important to review that information 6. To test that the target has been punctured, feel for
with the patient and answer any questions. differences in resistance or the presence of a capsule
6. Obtain informed consent. This consent should indi- as the needle is advanced. Move the syringe pistol
cate that name of the patient who is having the aspira- slightly from side to side. If the mass has been pene-
tion, the name of the doctor performing the aspiration trated, it will move under the palpating fingers unless
and a listing of discussed complications. Generally it is completely fixed to underlying or surrounding
there are two potential though rare complications tissues.
that need to be presented to the patient, bleeding and 7. Apply suction to the aspirating syringe, about one-
infection. These are very general complications that third of the total length of the syringe.
can occur anytime the skin surface is breached with 8. With suction held steady, the needle is moved back
any object, needle, knife, etc. The standard consent and forth within the lesion, using short, rapid strokes,
form that is used for small procedures is usually suffi- and within the same or nearly the same original
ciently generic that a specific one for FNA need not be direction of the needle. The needle sampling should
designed. Following current patient safety guidelines describe a narrow cone with the apex of the cone at
of the Joint Commission for Accreditation of Health the junction of the hub of the needle and the needle
Care Organizations (JACHO), it is important to use itself.
two identifiers for a patient, name and medical record 9. During the actual aspiration, watch carefully the junc-
number being preferred. It is also important to review tion of the needle and the tip of the syringe for the
with the patient and an assistant, usually a nurse, the appearance of any specimen. Absolutely critical for
procedure site, correct side if a lateralized site (right obtaining high-quality aspirates is to keep the speci-
breast, left breast), a then take a short “time out” to men within the needle. Aspirating excessive blood or
again review all of the above. Documentation of these fluid immediately dilutes the cellular
steps is obviously important. components of the biopsy.
10. When the specimen first appears at the junction of
While for many years the author did not obtain formal writ- the syringe tip and needle, release the trigger of the
ten informed consent, in compliance with the current interest of syringe pistol, allowing the vacuum in the syringe
maximizing patient safety and for a fully documented record of to equate to normal. A sample may not always be
the FNA procedure, informed consent is now obtained in every observed in the hub of the needle even after 10 or 12
case within the clinics and for all deep aspiration biopsies and rapid strokes of the needle within the tumor. This is
for any FNAs performed on children (minors). Patients already not an indication of an unsuccessful aspiration. When
admitted to the hospital have signed a general consent form for approximately this number of excursions of the
diagnosis and treatment, which the author believes is satisfactory. needle has been completed, if no sample is seen, stop
Some practitioners may feel that written informed consent is not the aspiration by releasing the trigger of the syringe
required for superficial aspiration biopsy that verbal consent will pistol, allowing the vacuum in the syringe to return to
suffice. If only verbal consent is obtained it should be thoroughly normal.
documented in the patient’s record. Obtaining permission for 11. Now that there is no longer any vacuum in the
aspiration biopsy should be in conformity with clinic and or hos- syringe, gently and slowly withdraw the needle from
pital policy. It is best to review this matter with your office of risk the mass.
management or your medical liability insurance carrier, or both. 12. Immediate pressure is applied to the puncture
The best defense against problems, however, is a caring attitude site with a sterile gauze pad. This is best done by
toward patients and taking time to fully inform them of what you an assistant (nurse) since the aspirator will be
are doing and how it may help resolve their medical problem. ­immediately occupied with preparing smears and
otherwise triaging the sample
Performing the Aspiration It is very important not to withdraw the needle from the
Listed here are the steps for actual performance of the aspiration lesion with any vacuum pressure still in the syringe. If there is
biopsy. vacuum present in the syringe, or the needle is withdrawn with
vacuum applied, the small aspiration sample will be pulled up
1. Grasp the lesion to be biopsied with one hand, into the syringe. Extracting the aspiration biopsy from the barrel
most often with two fingers, or push the mass into a of the syringe is quite difficult, the cellular material begins to
­position where it seems fixed and stable. dry almost immediately, and a good quality specimen is com-
2. Prepare the skin with an alcohol sponge as you would promised or could be irretrievably lost. Some aspirators pro-
for a venapuncture. The author currently swabs the pose that a small amount of balanced salt solution or heparin
skin twice with two different alcohol sponges. be added to the syringe, the former to avoid drying artifacts, the

584
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Fine-Needle Aspiration Biopsy Techniques

A B

Fig. 20.1  (A) Advance the plunger of the syringe to express a small drop of the sample, approximately 2–3 mm in diameter, onto the center of a glass slide.
This may be performed over a series of slides, or using one slide at a time followed by preparing the smear. (B) Invert another plain glass slide over the drop;
as the drop spreads, the two slides are pulled apart horizontally in a single gentle motion.

latter to prevent or inhibit clotting. The author has found that Many masses aspirated by image guidance are not homo-
both solutions dilute the specimen and destroy cytologic pat- geneous. Sampling the middle of a necrotic tumor will not
terns when preparing smears. lead to a correct or even satisfactory diagnosis. The presence of
A cyst may be encountered in aspirating a mass. This is not the pathologist to immediately prepare smears and report the
an uncommon occurrence when aspirating a thyroid or breast non-diagnostic nature of the smears should lead to a more pre-
nodule. The cyst should be evacuated as completely as possible. cise placement of the needle at the edge of a lesion to obtain
When a cyst is encountered, the author moves the needle in a definitive diagnostic material that is better preserved. This
wider cone while pressing on the mass to help evacuate it as ­collaborative type of service helps significantly in obtaining
completely as possible. It is useful to inform the patient that high-quality ­specimens and adequate samples for any ancillary
a cyst has been found and that it may take a moment longer studies that need to be performed.
to evacuate it, keeping in mind the patient’s level of comfort Developing the necessary confidence to perform FNA takes
with the procedure. When a cyst is aspirated, the author with- practice. Some experience and confidence can be obtained by
draws the needle with some vacuum in the syringe in an effort the performance of aspiration biopsies on both cadavers and
to remove the last drop of fluid from the mass and collapse the surgical specimens. The mechanics of handling the syringe gun
walls together using the palpating hand. Excessive pressure is not and the several steps involved in the aspiration biopsy will
necessary to accomplish this. The most important point when become automatic. Smear preparation, which is discussed next,
aspirating a cyst is to re-examine the patient for any residual is also an important part of any practice; both to gain facility
mass and to re-aspirate any lesion that remains. This may help at this important technique and to collect smears made from
to ensure that a true neoplasm is not overlooked, particularly a normal tissues as well as neoplasms both benign and malignant
metastatic tumor that has undergone cystic degeneration. and even non-neoplastic conditions. This collection of smears
As described above, the aspiration technique for deep lesions forms an excellent reference source for comparing with patient
is the same, following the administration of local anesthesia, specimens.
through the chest or abdominal wall to the level of the pleura or
peritoneum. Anticipating that multiple aspirations will often be
made of the same mass, radiologists will place a guide needle,
Smear Preparation
usually 18-gauge, through the skin, subcutaneous tissue, and 1. Immediately after completing the aspiration biopsy,
muscle and close to the surface of the target for biopsy. The cor- quickly remove the needle from the syringe; then pull
rect position of the guide needle is then checked by imaging. back on the syringe pistol to fill the syringe with air.
The aspirating needles of 21 and 22 gauge or needles for micro- 2. Reattach the needle; place the needle near the center
core biopsy are then inserted through the guide needle. Modern and touching the surface of a plain glass slide.
imaging techniques allow the actual depth of the target to be 3. Advance the plunger of the syringe, which will express
determined quite accurately. A stop is placed on the aspiration a small drop of the sample, approximately 2–3 mm
needle so that the depth of the mass is correctly determined. in diameter, onto the slide (Fig. 20.1A).
After placement of the aspiration needle through the guide nee- 4. Quickly continue this procedure over a series of five
dle, its position may also be rechecked by imaging before the to six slides.
actual aspiration sample is obtained. Because of the motion of 5. Invert another plain glass slide over the drop; as it
respiration may affect the correct placement of the aspirating spreads from just the weight of the slide, pull the two
needle, particularly for lung lesions, the patient is asked to hold slides apart horizontally in a single gentle motion
their breath when the aspirating needle is inserted into the mass (Fig. 20.1B).
and the sample obtained. This maneuver also reduces trauma to 6. As an alternative, when the drop spreads in a circu-
the pleura, which in turn lowers the risk for pneumothorax. lar fashion, again from the weight of the slide, pull

585
 PART TWO Diagnostic Cytology

A B

C D

586
E F
 20
Fine-Needle Aspiration Biopsy Techniques

Fig. 20.2  (A) A drop of biopsy material is placed toward one end of a glass slide approximately 5 mm in diameter. The slide with the drop of biopsy material
is held in the left hand. Take a second slide with the right hand, bringing the edge of that slide up to the drop. The drop of biopsy begins to spread along the
edge of the slide in the right hand. (B) As it does, begin to push the edge of the slide in the right hand toward the end farthest from the drop, but only about
halfway down the slide in the left hand. This procedure is similar to making a blood smear. (C) When the halfway point of the slide in the left hand is reached,
lift the spreader slide (in the right hand) straight up off the slide in the left hand. (D) Immediately tilt the smear slide in the left hand away from the leading
edge of that smear. Note that the blood runs back toward the point where the drop of sample was placed originally. (E) Next begin smear preparation with
the lateral clean edge of the slide in the right hand (spreader slide), but only from the leading edge of the slide in the left hand. (F) Tissue particles that may
be present will be concentrated in that part of the smear prepared from the leading edge of the slide in the left hand.

the two slides apart vertically (compression or pop 4. Note that the drop of biopsy sample spreads along
smears). The pop smear method is useful when the the edge of the slide exactly as it would if one was
specimen is relatively fluid versus semisolid (often making a peripheral blood smear. As this drop of
the situation with thyroid aspirates). sample spreads along the edge of the slide, begin to
7. Repeat the above procedure for all slides; fix some of push the edge of the slide in the right hand toward
the slides immediately in 95% ethyl alcohol, or other the end farthest from the drop of sample, but only
suitable fixatives, depending on stain preferences, as about halfway toward that end of the slide in the left
you make each smear. hand. (This same method would be used in making a
8. Allow unfixed smears to air-dry. blood smear.) (Fig. 20.2B,C)
5. Reaching the halfway point as described in step 4, lift
It is very important to place the bevel of the needle against the the spreader slide (in the right hand) straight up off
slide as the sample is expressed so that there is no air gap between the slide in the left hand.
the end of the needle and the surface of the slide. Splattering the 6. Tilt the smear slide in the left hand immediately away
biopsy over the surface of the slide is prevented, as is excessive from the leading edge of that smear; note that the
air-drying of the aspirate before smear preparation and fixation blood runs back toward the point where the drop of
in alcohol. After practicing smear-making methods, it becomes sample was placed originally (Fig. 20.2D).
possible to place nearly all the aspirated material, normally 4 7. Next, begin smear preparation with the lateral clean
or 5 drops in a 3.8-cm (1½-in.) needle, over a series of four to edge of the slide in the right hand (spreader slide) but
six slides; then actual smear preparation can begin. Utilizing only from the leading edge of the specimen, from the
the rapid Papanicolaou method of Yang, all smears may be air- slide in the left hand (Fig. 20.2E).
dried.46 The most important point in preparing smears is to have 8. Tissue particles that may be present will be
the smear occupy only a small area of the slide. With good smear ­concentrated in that part of the smear prepared from
technique, a tissue-like pattern is created, particularly when the the leading edge of the slide in the left hand (that
aspirate is from a neoplasm. The area of the smear that must be portion of the specimen that the blood ran away
reviewed by the cytopathologist or screened by a cytotechnologist from when the slide in the left hand was tilted)
is reduced, enhancing potentially important diagnostic features. (Fig. 20.2F).
Some aspiration biopsies are inevitably diluted by excessive
blood or fluid. The majority of aspirates from non-neoplastic The method of smear preparation described above is difficult
goiter of the thyroid are largely composed of colloid mixed with and requires substantial practice and dexterity to achieve reason-
some blood. These smears contain relatively few follicular cells. able results. It defeats clinicians and radiologists. If a specimen
Inexperienced radiologists and/or clinicians often provide sam- is largely fluid, then it is entirely appropriate and more practical
ples excessively diluted by blood. Their concept is more sam- to use standard cytologic techniques as one would for handling
ple, be it blood or fluid or both, is better. The opposite is the any other fluid submitted to the laboratory. The author’s prefer-
case. It is difficult to make good smears from specimens largely ence is for cytospin preparations. Use of filters has disappeared,
composed of blood. Receiving a tray or two of splattered bloody as have cell spreads of buttons of material obtained from some
smears where a concerted attempt has been made to cover the fluids after centrifugation. Breast cyst fluid that is aspirated may
entire slide is a waste of effort. Some cells, usually in no recog- be submitted for cytologic examination in this manner, but if
nizable pattern and few in number, may be present most often the fluid is clear and yellow, it can safely be discarded without
at the edges of some of the smears. They consist mostly of single cytologic examination. Cyst fluid from breast aspirations that
cells. In dealing with bloody or diluted aspirates, the follow- is cloudy and/or discolored should be examined cytologically.
ing steps may help in concentrating whatever cellular material Cytospin preparations are also used in the author’s laboratory
might be present. because immunohistochemical staining can be performed on
them as needed.
1. Place the drop of biopsy sample on the slide as When radiologists or other clinicians perform image-guided
described previously; because of the liquid nature of aspirations or intraoperative aspiration biopsies are obtained by
the aspirate, this drop will spread of its own accord surgeons, they often are composed of large volumes of blood
and probably be 5–10 mm in diameter. pulled into the barrel of the syringe. The best method for han-
2. Hold the slide with the drop of biopsy in the left dling that entire portion of the sample within the syringe is to
hand. place it in 10% neutral buffered formalin and process it as a
3. Take a second slide in the right hand, bringing the cell block, cutting multiple levels (see later). Only that portion
edge of that slide up to the drop and at about a of the aspirate remaining within the needle should be used to
30°–45° angle (Fig. 20.2A). prepare smears.

587
 PART TWO Diagnostic Cytology

Even a few milliliters of blood drawn up into the syringe Smears to be stained by a rapid hematoxylin and eosin ­benefit
­ arrel can result in the rapid formation of a clot. Clotting of
b from immediate fixation by immersion for a few seconds in
blood greatly interferes with good smear preparation. Whatever equal parts of 50% ethyl alcohol and 10% neutral buffered for-
cellular material may be present becomes entrapped within malin. This brief fixation also improves staining of routine fro-
the clotted blood. The cells cannot be spread satisfactorily on zen sections. When staining with a quick hematoxylin and eosin
the slide, often destroying any useful diagnostic pattern to the method, in the rinsing steps use hot (tap, not boiling) water.
smear. The presence of the blood with entrapped cells also This improves the overall quality of the slides and ­sharpens cell
results in heavy and uneven staining, making cells appear much detail.
more hyperchromatic than they would be if spread evenly. Cytospin preparations for immunohistochemistry are pre-
Samples for cell blocks may be handled by a variety of pared by rinsing either a separate aspirate from the lesion or a
methods: (1) simple centrifugation and the addition of 10% portion of the aspiration biopsy into balanced salt solution. A
neutral buffered formalin to the resulting cell button, (2) addi- separate aspirate for cytospins is preferable. Cytospins may be
tion of thromboplastin or bacterial agar to the cell button, or either air-dried or fixed in 95% ethyl alcohol. The author prefers
(3) the commercially available cytoblock system. The author air-drying. Direct smears or cytospins either air-dried or fixed
has found the cytoblock system to be most satisfactory (see to be used for immunohistochemistry may be stored in a deep
Appendix). With any cell-block method, it is important to freezer for up to 4 weeks. Aspiration samples either fixed or deep
work closely with the histotechnologist so that the blocks are frozen are also suitable for molecular diagnostic methods and
trimmed carefully to cut ribbons of thin sections of the block. may be stored as described above.
Some of these histologic slides should be stained initially Both personal preference and experience seem to dictate
(e.g. slides that are odd numbered), while saving others to be which stain or stains will be used on aspiration biopsy smears.
stained later if needed or for the application of special stains. Cytopathologists with an orientation toward surgical pathol-
Cell-block sections are also more consistent for the applica- ogy may prefer a rapid hematoxylin and eosin stain. Those with
tion of immunohistochemistry, with the exception of cases experience in cytopathology most often prefer the Papanicolaou
of lymphoproliferative diseases, for which cytospins provide stain. Individuals influenced by a hematologic background or
good consistent preparations if enough cells are obtained from trained in the aspiration biopsy clinic at the Karolinska or in
the FNA. other Scandinavian countries seem to like the Romanowsky
The tip design of the Franseen-style needle seems to result in stains, May-Grünwald-Giemsa, straight Giemsa Wright’s stain,
the procurement of microcores from the aspiration procedure. It or, in the United States, Diff-Quik.80,81 The basic stain discov-
is usually necessary to dislodge these very small cores by insert- ered by Romanowsky is azure A. This compound is unstable and
ing the stylus into the needle, then pushing them out onto a is continually oxidized to azure B, which is the actual staining
plane glass slide. Do not attempt to smear these small cores. substance. This type of stain results in a metachromasia of any
That will only result in distortion of the cells and poor qual- stromal elements present with epithelial or other types of cells
ity material from which to make an interpretation. To prepare seen in contrast to that stroma. In addition to this sharply dis-
some smears, at least in an effort to provide a provisional inter- tinguishing metachromasia, the major advantage of this stain
pretation, gently roll the microcore over a small area of the slide. is its rapidity, which can lead to an immediate interpretation
Then the core should be placed very quickly in suitable fixative, ­following the aspiration biopsy. The quality of the aspirate and
10% neutral buffered formalin, and processed as a small biopsy. the smears can also be checked. Repeat aspiration biopsies, if
Because these tissue cores may be quite slender, it is equally necessary, can be obtained for both diagnosis and special stud-
imperative to work closely with a knowledgeable histotech- ies as required from the preliminary evaluation.
nologist to obtain good tissue sections and not destroy much The Diff-Quik stain is a three-step procedure. It takes less
of the tissue when trimming the paraffin block. Some of these than 20 seconds to obtain a stained aspiration smear with this
microcores can be difficult to see when embedded in a paraf- stain. Table 20.1 is a general comparison of the properties of
fin block. Use of a stain, such as mercurochrome, may help the air-dried versus wet-fixed smears. Table 20.2 presents the fea-
histotechnologist see them more clearly within the block. These tures emphasized by the Romanowsky stains compared with the
microcores require even more skill in histology than ­handling conventional Papanicolaou stain as outlined originally by Orell
the traditional somewhat larger needle core biopsies of liver or and colleagues.80
kidney. It has been the author’s experience that the conventional
Papanicolaou stain results in some significant loss of cells from
smears. Part of this occurs during fixation, even with spray
Fixatives and Stains ­fixatives, and additional cells are lost during the staining proce-
dure. In 1995 a very rapid Papanicolaou stain was developed and
Smears that are air-dried are stained by a Romanowsky method reported by Yang.46 For this staining method, aspiration biopsy
or one of a number of variations. The author prefers the com- smears are allowed to air-dry. The smears are then rehydrated
mercially available Diff-Quik stain. This stain is a three-step with normal saline as the first step of the staining procedure. Next
process composed of methyl alcohol, which is the fixative, fol- is fixation by alcoholic formalin, a mixture that is 65% ethanol
lowed by eosin Y and then azure A. Smears spray-fixed with one and 4% formalin. Details of this staining method are provided
of a number of commercial products available or wet-fixed in in the Appendix. Total fixation and staining time is usually only
95% ethyl, methyl, or isopropyl alcohol are stained with Papan- 90 seconds. It has been helpful in some cases to have the rapid
icolaou stain or its modifications. As noted for the rapid Papani- Papanicolaou-stained smears to compare cytologic features
colaou stain all smears may be air-dried.46 When using spray with air-dried Diff-Quik-stained smears (Figs. 20.3 and 20.4).
fixatives, it is important to allow those smears to dry at least ­Problem cases may be more quickly resolved. An added advan-
1 hour prior to staining. tage is that clinicians performing their own ­aspiration biopsies

588
 20
Fine-Needle Aspiration Biopsy Techniques

Table 20.1  Comparison of Air-Dried and Wet-Fixed Smears

Wet-fixed smear
Air-dried smear (Papanicolaou
(­Romanowsky stain) stain)
Dependence on Strong Moderate
smear technique
Dry smear Good fixation Drying artifacts
­common
Wet smear Artifacts common Good fixation
Tissue fragments Cells poorly seen, Individual cells seen
heavy background clearly
staining
Cell and nuclear Exaggerated, differ- Comparable to tissue
area sections ences enhanced
Cytoplasmic detail Well demonstrated Poorly demonstrated Fig. 20.3  Aspiration smears from solitary thyroid nodule. Clusters
and overlapping nuclei and nuclear enlargement and elongation suggest
Nuclear detail Different pattern from Excellently demon-
papillary carcinoma. Nuclear grooves and intranuclear inclusions are not well
Papanicolaou stain strated
seen (Diff-Quik × MP).
Nucleoli Not always discernible Well demonstrated
Stromal Well demonstrated Poorly demonstrated
­components and often differentially
stained
Partially necrotic Poor definition of cell Good definition of
tissue detail single intact cells

Table 20.2 Comparison of Staining Features between Romanowsky Stain

and Conventional Papanicolaou Stain

Features
Features ­emphasized ­emphasized by
by ­Romanowsky stain Papanicolaou stain
Epithelial Mucin, intracellular, or Squamous
extracellular, colloid ­differentiation/
(thyroid); secretory keratinization;
granules (prostate); lipo- ­oncocytes (salivary
fuscin granules (seminal gland tumors); Fig. 20.4  Aspiration smears from solitary thyroid nodule. Same case
as in Fig. 20.3, rapid Papanicolaou stain, available 90 seconds after the Diff-
vesicles); lipid vacuoles; psammoma bodies.
Quik stained slides are ready for review. Note the same basic morphology
fire flares (thyroid); bare
of the cell groupings, suggesting papillary carcinoma. Good examples of
bipolar nuclei (benign
intranuclear inclusions and nuclear grooves are easily seen, confirming the
breast); bile plugs; initial impression from the Diff-Quik-stained smear of papillary carcinoma of
basement membrane the thyroid (Rapid Papanicolaou × MP).
globules (adenoid cystic
carcinoma); amyloid.
Lymphoid Cytoplasmic basophilia; Nuclear outline; enhance the diagnostic capabilities. Immunoperoxidase stain-
lymphoglandular bodies; nuclear chromatin ing can be performed directly on smears, on microcore–cell
hematopoietic cells; lipid pattern; nucleoli. block material, and on cytospin preparations of aspirates col-
vacuoles. lected in balanced salt solutions or RPMI, a cell transport media.
With the use of direct smears, background staining may be sig-
nificant, but if the cells in question are staining intensely, this
represents a positive reaction for the antibody in question. Iden-
do not have to consider the details of fixation of smears. Rather, tification of a terminally differentiated protein, such as gastrin
the laboratory can decide which smears to stain by this rapid or prostatic-specific antigen, is quite reliable on direct smears.
Papanicolaou method and which to stain using other stains. Background staining can be ignored and is likely from the tar-
get protein displaced by rupture of the cells during smearing.
The basic differential between lymphoma and undifferentiated
Ancillary Techniques and Applications carcinoma is also quite reliable when immunocytochemical
staining with CD45 versus a cytokeratin cocktail is applied to
The use of nearly all of the special techniques that have evolved direct smears. To obtain a clean background and in cases of lym-
for application to tissue specimens have been applied to aspira- phoproliferative disease, cytospin preparations (see Appendix)
tion biopsies and other types of cytologic samples.31 They may usually ­provide a good differentiation of positive and negative

589
 PART TWO Diagnostic Cytology

staining of various types of lymphoid cells and may be used to

determine clonality.
Complications of Fine Needle
Aspiration Biopsy
Organization of the Aspiration Biopsy Service A comprehensive review and discussion of the complications
of fine-needle aspiration biopsy has been reported by Powers.45
A pathology-based aspiration biopsy service may be organized A number of case reports and reviews have appeared since the
as part of different practice settings—hospital, independent lab- report of Powers.82,83 However, many of these reports are poorly
oratory, or free-standing clinic. Abele and Miller have detailed documented, specifically with reference to needle size. After
their experience of evolution from a hospital-based service to a thoroughly studying the literature, with restriction of FNA needle
free-standing clinic.47 It is important to have dedicated person- size of 22 gauge and thinner, and estimating the total number
nel familiar with the needs of patients in all of these practice of aspiration biopsies being performed, Powers determined that
venues. The utility of FNA must first be proved to clinicians. This the complication rate for FNA was approximately 0.03% of cases.
is best accomplished by providing on-demand procedures. That This percentage risk would make FNA one of the safest invasive
may include not only servicing hospitalized patients and those diagnostic procedures.45
in the hospital’s outpatient clinics but travel to attending physi- Complications of fine-needle aspiration biopsy of superfi-
cians’ offices. As the service grows it then becomes necessary to cial masses include needle track seeding; pneumothorax with
perform aspirations within designated hours. Sufficient space to breast, axillary, and supraclavicular masses, transient acute
examine patients is required. For the in-hospital clinic it should swelling (thyroid) hematomas, and histologic alterations. Tis-
be located close to the pathology department but should be sue alterations have occurred with aspiration biopsy of lymph
a separate designated area that is quiet and comfortable for nodes, thyroid, salivary gland, and breast. These have rarely
patients and with sufficient support staff to register patients, posed a problem in interpretation of the excised tissue.45 Tissue
provide assistance to the pathologist performing the aspiration, alterations also occur with core needle biopsies and have cre-
and take care of all clerical matters. ated some problems specifically with an initial interpretation
As described by Abele and Miller, physicians may then of intraductal carcinoma of the breast followed by subsequent
request that FNAs be performed in their office in preference to excision for the determination of invasion and/or the meaning
some location in the pathology laboratory.47 Fortunately aspira- of finding tumor in lymphovascular spaces. Some of these tissue
tion biopsy does lend itself to travel because there is a minimum alterations may be attributed to faulty technique by inexperi-
of equipment to transport. The major disadvantage is the travel enced clinicians performing the procedure.84 Often larger than
from office to office for single biopsies in even a modest-sized 22 gauge needles or microcore needles of 21 gauge are being
metropolitan area. However, this investment in time does pro- used in these cases, with vigorous attempts to obtain tissue cores
mote the use of aspiration biopsy, which if successful, leads to rather than traditional aspiration biopsy to prepare smears.85
the next phase, the establishment of a free-standing clinic. A Serious and sometimes life-threatening complications may
presentation to clinicians is an important marketing strategy for occur with aspiration of deep organs or masses. With ­transthoracic
aspiration biopsy to be accepted. aspirations, these include pneumothorax, massive hemorrhage,
There are a number of considerations in developing and air embolism, and cardiac tamponade. No cases of massive
planning a free-standing clinic. ­hemorrhage have occurred with 22-gauge or thinner needles.
1. Location Two cases of air embolism have been reported from aspiration
a. Convenience with 22- and 23-gauge needles, the latter also using a 19-gauge
b. Ground floor guide needle. A single case of tamponade has occurred with a
c. Within an established medical facility 22-gauge needle. The patient survived.45 Several cases of needle
d. Near offices of physicians referring the majority of track seeding of tumor have been reported with transthoracic
the patients FNA, but in 10 of 13 patients either the gauge was not given or
e. Perception that location is medically reputable the needle was 20 gauge or larger. Three cases were ­documented
2. Patient convenience where the aspiration biopsy was performed using a 22- or
a. Easy accessibility to public and private transportation 23- gauge needle, with one case also using a guide needle of
b. Obvious designated entrance 19 gauge.45
c. Ample parking off a major street Twelve cases of complications leading to death have resulted
d. Clinic personnel who can instruct patients how to following aspiration biopsy of the liver. Two of these occurred
reach the clinic from any location using 22-gauge needles. In a third case, the gauge is not given, but
3. The facility the needle is stated to be “fine.”45 Needle track seeding has also
a. Two examining rooms, one somewhat larger than been reported with transabdominal aspiration biopsy. Organs
the other involved include liver, pancreas, kidney, and a variety of miscel-
b. Some counters at waist level for standing laneous sites. Two of eight reported cases after liver FNA devel-
c. Examining table pointed toward outside windows oped subcutaneous tumor implant. A 22-gauge needle was used
and away from aspiration instruments and smear in these cases. Four of ten similar cases occurred with 22-gauge
preparation area needles used for aspiration biopsy of the pancreas. The implants
d. Space set aside for children were most often subcutaneous or dermal nodules, while one
e. Fixed bench for children, with collection of stuffed was a peritoneal nodule. In one of five cases after FNA using
­animals a 23-gauge needle there was a dermal tumor implant.45 Over-
f. Toy box with safe, appropriate toys all therefore the complication rate is quite low. Risk factors that
g. Interior decoration with home-like atmosphere should be considered that may influence the ­ development of

590
 20
Fine-Needle Aspiration Biopsy Techniques

complications following FNA are patient age and sex, presence cooperation is required between radiologists, endoscopists, and
of an underlying disease, and bleeding diathesis. Also exerting pathologists to achieve excellent results. Neither radiologists,
influence on the rate of complications are location, size, and endoscopists, nor other clinicians have demonstrated any exper-
depth of the mass; needle size; number of passes; and the level tise in handling the aspiration biopsies they obtain or in prepar-
of experience of the aspirator.45 ing other than bloody useless smears. They substitute placing
the aspirate in some preservative fluid, sending it to a pathology
laboratory, either locally or at some distance, with no modicum
Concluding Remarks of clinical information. This is a recipe for potential disaster.
Ultrasound imaging is not required to aspirate palpable masses
Fine-needle aspiration biopsy continues to expand its dimen- at any site, but sound aspiration technique, good information
sions in the diagnosis of a wide variety of diseases with a strong gathering skills, and excellent smear preparation is required. A
focus on neoplasms. Fundamental to its success, however, are recent paper by Hehn and colleagues concluded that overall,
the basics of gathering accurate and complete clinical history, FNA for the diagnosis of lymphoma was not useful.86 A lym-
technical competence in obtaining the aspirate, preparation phoma specialist reviewed 470 medical records of new patients.
of initial smears and their evaluation, and subsequent obtain- Twenty-one percent of the patients had undergone a total of 115
ing and triaging additional samples as needed for the appli- fine-needle aspiration biopsies. All of these aspirates were taken
cation of appropriate new technology to enhance diagnostic by clinicians and were sent to 32 different pathology labora-
accuracy. This approach cannot be left to chance and is only tories and interpreted by more than 70 different pathologists.
really within the purview of pathologists. While they are few Immunophenotyping was performed in about one-third of the
within the United States, the pathology-based aspiration biopsy cases.86 Based on this approach the conclusions of this paper are
services, either within academic or private practice settings or inevitable. American pathologists will have to decide whether
free standing FNA clinics, continuously achieve the best results they wish to be involved in FNA and control the application of
with FNA. Imaging has enhanced the ability to find and biopsy sophisticated technology as it evolves, or abdicate their respon-
deep and sometimes incidental lesions. However, a very close sibilities to others.

Appendix

Equipment

1. Cameco Syringe Pistol. 20.0 and 10.0 mL size. Street, London, Ontario, N6A 3S6, Canada. Phone:
Available from Mortonmedical. Phone: 011 44 (0) 519 642 0424; fax: 519 642 0426; e-mail: dynabc@
208 8711444; fax: 011 44 (0) 208 8779888; e-mail: allstream.net; website: http://www.dynamedical.
[email protected]; website http:// com/
www.mortonmedical.co.uk 8. ROTEX II needle 0.55 mm., 15 and 20.0 cm length
2. Aspir-Gun. Available from Nayfeld Corporation. (used primarily for hamartomas of the lung).
Phone: 415 775 7117; fax: 415 775 6436; website: ­Available from Ursus Konsult AB, Grev Turegatan 2,
http://www.nayfeld.com/ S-114 35, Stockholm, Sweden.
3. Inrad Aspiration Syringe Gun (Tao Gun). Available
from INRAD. Phone: 1 800 558 4647; fax: 616 301 Staining techniques
7799; e-mail: [email protected]; website:
http://www.inrad-inc.com/
4. 20- and 10-mL disposable plastic syringe with LuerLok
1. Papanicolaou Stain
Tip. Available from Becton Dickinson, 1 Becton Drive, Any of the modifications available in most laboratories are
Franklin Lakes, NJ 07417. Phone: 201 847 6800; web- satisfactory. We prefer Gill’s Hematoxylin, available from Poly-
site: http://www.bd.com/ sciences, Inc., 400 Valley Road, Warrington, PA 18976. Phone:
5. 22-, 23-, and 25-gauge, 0.6- to 1.0-mm external 800 523 2575; fax: 800 343 3291; e-mail: info@polysciences.
diameter disposable needles, 3.8 and 8.8 cm long, com; website: http://www.polysciences.com. Staining times may
with or without mandrin. (Used for most aspirations need to be adjusted for variations in thickness of smears. We also
of palpable lumps.) Available from Becton Dickinson use Gill’s modified OG-6 and EA for our Papanicolaou stain,
(see above). also available from Polyscience.
6. Aspiration biopsy needles 15 and 20 cm (6 and 7 in.)
long, 21 and 22 gauge, with or without mandrin. (For
biopsy of lung, transabdominal and pelvic masses,
2. Rapid Papanicolaou Stain46
and prostate.) Available (as well as Chiba- and This is a 90-second staining protocol that has proved quite
Franseen-style needles) from Cook Co. Inc., PO Box ­effective in producing high-quality Papanicolaou-stained
489, Bloomington, IN 47402; website: http://www. smears and at the same time reducing or eliminating blood in
cookmedical.com/ the background of an aspiration smear. Smears are made in the
7. Franseen-style needle, 22 gauge, 3½ in. with man- usual fashion and allowed to air-dry. The staining protocol is as
darin. Available from Dyna Medical 843 Wellington ­follows:

591
 PART TWO Diagnostic Cytology

a. Normal saline 30 seconds tap water ­ following immersion in solution I and before dip-
ping the slide in solution II.
b. Alcoholic formalin* 10 seconds
c. Water 6 slow dips (at speed
of 1 dip/second) 4. Modified May-Grünwald-Giemsa Stain
d. Richard-Allen hematoxylin 22 slow dips
May-Grünwald stock stain 1.0 g eosin-methyl blue in 1000 mL
e. Water 6 slow dips methyl alcohol
f. 95% ethanol 6 slow dips Giemsa stock stain Add 1 g Giemsa powder to 66 mL
g. Richard-Allen Cyto-Stain† 4 slow dips glycerin. Incubate at 37ºC for 3 hours,
mixing occasionally. Add 66 mL
h. 95% ethanol 6 slow dips
methyl alcohol to the incubated stain.
i. 100% ethanol 6 slow dips Store in the refrigerator.
j. Xylene 10 slow dips May-Grünwald working To 40 mL of stock stain add 20 mL
Mount in mounting medium and cover slip. stain methyl alcohol in a Coplin jar.
*Three liters of alcoholic formalin is conveniently prepared by combining 300 mL Giemsa working stain Add 45 mL of Giemsa stock stain
of 38–40% formaldehyde, 2053 mL of 95% ethanol, and 647 mL of distilled water. to 45 mL of distilled water in a
This makes a mixture that is 65% ethanol and 4% formaldehyde. This is a potent Coplin jar.
fixative, and smears should not be transported in it. Prolonged immersion in this
fixative will adversely affect the cytomorphology.
†Richard-Allen Cyto-Stain is an alcoholic mixture of orange G, eosin Y, light green,
and aniline blue. Both this stain and Richard-Allen hematoxylin 2 are available from Staining Procedure
Richard-Allen Scientific, 4481 Campus Drive, Kalamazoo, MI 49008; phone: 800 522 Immerse the air-dried aspiration smears in May-Grünwald work-
7270; fax: 269 372 2809; website: http://www.rallansci.com. There are small-volume
portable staining racks that easily accommodate this number of solutions and can
ing stain for 15 minutes. Rinse gently in tap water. Immerse the
be transported to any site (e.g. radiology department). The Richard-Allen dyes are smears in Giemsa working stain for 15 minutes. Rinse gently
quite stable but should be filtered at least weekly. All other solutions need to be with tap water. Allow to air-dry. Dip in xylene for 10 seconds and
changed daily. mount in Permount.
Prepare May-Grünwald working stain fresh once per week.
Prepare Giemsa working stain fresh daily. The stock Giemsa
stain is good for 6 months if refrigerated. The stock May-
3. Diff-Quik Stain Set ­Grünwald stain is good indefinitely and does not require refrig-
This commercial stain kit, a modified Wright stain, is a three- eration.
solution, three-step method that is both fast and practical.
It provides good cell detail and identifies stromal fragments
by their metachromasia. This stain is comparable to the May-
5. Hematoxylin-Eosin Stain
Grünwald-Giemsa and the Wright-Giemsa stains, but it is much Eosin Y solution
quicker. Fixation is with methanol containing 1.8 mg/L triaryl- Harris hematoxylin
methane dye, 100% pure dye content. Solution I is buffered Dilute ammonium hydroxide
eosin Y. Solution II is a buffered solution of thiazine dyes, meth-
ylene blue, and azure A. Azure A undergoes slow but constant Add one drop of concentrated ammonium hydroxide to 100 mL
oxidation to azure B, which is the actual staining solution of the of distilled water.
original Romanowsky method. The stain kit is available from
VWR; phone: 800 932 5000; website: http://www.vwrsp.com, Staining Procedure
and also available from Dade Behring Inc., 1717 Deerfield Rd., Use on air-dried aspiration smears.
Deerfield, IL 60015; phone: 800 241 0420 or 847 267 5300;
website: http://www.dadebehring.com. a. Absolute ethyl alcohol 1 minute
b. 95% ethyl alcohol 1 minute
Staining Procedure
c. Tap water several dips
The air-dried smears are dipped for 5 seconds (five dips) in,
fixative, solution I and solution II, respectively. The excess stain d. Harris hematoxylin 2 minutes
is drained from the slides between solutions. Following the e. Tap water several sips
last staining solution (II), the slide is rinsed with water and f. Dilute ammonium hydroxide 1 to 2 dips
either allowed to dry or examined wet. When the smear com-
pletely dries, it may be made permanent by immersing it in g. Eosin Y 30 seconds
xylene for several seconds and mounting it with Permount and h. Tap water several dips
a coverslip. Staining times may need modification depending i. Tap water several dips
on the thickness of the smear and the age of the stain. After
j. 95% ethyl alcohol several dips
completing the Diff-Quik stain examine the slide grossly. The
surface should have a purple-blue color, not brown or orange. k. 95% ethyl alcohol several dips
If the latter color is present, the slide is understained. The smear l. Absolute ethyl alcohol several dips
should be dipped a few more times in solution II, and then m. Acetone several dips
rinsed in tap water again. It is quite difficult to overstain with
n. Xylene 1 minute
Diff-Quik. The author also prefers to rinse the slide briefly in

592
 20
Fine-Needle Aspiration Biopsy Techniques

16. Reclose the cytoblock cassette and place flat side up

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

593
 PART TWO Diagnostic Cytology

a series of blocking steps to quench endogenous peroxidase

Materials and nonspecific protein binding, the primary antibody is then
1% saponin (weight per volume (W/V)) located by the application of a horseradish peroxidase-labeled
1 g saponin, 0.2 g sodium p-hydroxybenzoate (preservative) polymer, which is conjugated with secondary antibodies and
and 99 mL distilled water monoclonal or polyclonal specific. Following the application
3% calcium gluconate (W/V) of the HRP polymer the specific antibody and conjugated poly-
3 g calcium gluconate, 0.2 g sodium p-hydroxybenzoate mer enzyme complex is then visualized utilizing a precipitated
(­preservative) and 97 mL distilled water enzyme product (DAB or DAB+). All steps of the actual staining
procedure take place at room temperature and on an automated
Prepare solutions listed above in beakers. Mix thoroughly. immunostainer manufactured by Dako Corporation.
Filter into clear brown bottles; date and label. Expiration for
these solutions is 2 weeks. Collection and Handling of Specimen
1. Tissue sections are fixed in 10% NBF for 6 to 24
Method hours. Tissues are routinely processed to paraffin
1. Pour sample into designated centrifuge tube, blocks and sectioned at 3 μm, and sections are placed
­leaving sufficient space for addition of saponization on charged glass slides. Sectioned slides are stored at
­solutions. (See steps 2–4.) room temperature in a desiccator until the test is to
2. If specimen is grossly bloody, add 2 disposable be performed.
pipettes full of 1% saponin and mix with a vortex 2. Cytospins, aspirates, and smears are to be prepared
motion for 30 seconds. Let sit for an additional 30 within 12 hours of specimen collection. Slides are
seconds. Coloration and/or transparency of the sam- then fixed in anhydrous room temperature acetone
ple should change noticeably. for 15 minutes. Slides are air-dried for a minimum
3. Add 3 disposable pipettes full of 3% calcium gluco- of 30 minutes and may be kept in a desiccator up to
nate and mix with a vortex motion for 30 seconds. ten days before performing an immunohistochem-
4. The saponin and calcium gluconate must always be istry test. Cytospins, smears, and aspirates are then
used in a 2:3 ratio. The actual amounts may be varied post-fixed in 50% ethanol for 1 minute just prior to
according to the amount of blood present in each starting staining protocol.
specimen. It is feasible to use only 2 drops: 3 drops if
blood is present only in minute amounts. Adequacyof Specimen
5. Proceed to centrifugation and cytospin preparation. 1. Any well-fixed tissue that has been paraffin-processed
6. If the presence of blood is detected only after initial and adequately sectioned onto a charged slide is con-
centrifugation, saponin may still be used. Decant sidered acceptable. Paraffin blocks or unstained/H&E
supernatant, vortex suspension, and add the solu- stained slides may be submitted.
tions in a ratio 2 drops: 3 drops, respectively. Proceed 2. Cytospins, aspirates, and/or cell smears that have
with preparation of cytospins. been prepared within 48 hours of specimen col-
lection are considered acceptable. Specimen prepa-
rations are acceptable up to 1 week after initial
Supravital Stain preparation if stored at room temperature in a
desiccator. Four slides with cell or tissue preparations
Toluidine Blue should be submitted for each antibody requested.
0.5g toluidine blue
20mL 95% ethyl alcohol Reagents
80mL distilled water 1. Acetone (Fisher)
Dissolve toluidine blue in alcohol; add distilled water, filter, 2. Xylene (Cardinal)
and store in dark bottle in the refrigerator. 3. Ethanol (Richard Allen)
4. 10× wash buffer pH 7.5 (Dako S3006)
Immunostaining of Cytospins Using the Dako 5. DAB substrate chromogen (Dako K3466)
6. Biotinylated goat anti-mouse and anti-rabbit antibodies
Autostainer 7. Envision plus–Peroxidase conjugate (Dako K4007 for
mouse antibodies or K4010 for rabbit antibodies) or
Principle of Method87 Envision plus Dual link polymer (Dako K4061)
This test utilizes a manual procedure based upon an enzyme- 8. Normal goat serum (Dako)
labeled horseradish peroxide polymer (HPR) complex meth- 9. Hematoxylin (Gill’s III, Sigma)
odology. Paraffin sections are deparaffinized then rehydrated. 10. 10× Dako target retrieval solution (Dako S1699)
Cytospins, aspirates, and smears are fixed in an anhydrous 11. 10× Dako target retrieval solution, pH 9 (Dako
acetone and post-fixed in 50% alcohol (for cytospins direct S2367)
smears). The tissue or cell preparations are then treated with 12. Antibody diluent w/background reducing compo-
a series of immunologic and/or related steps. This test utilizes nents (Dako S3022)
a primary antibody that is commercially available and which 13. 10% neutral buffered formalin
had been titered in a known positive and negative control mul- 14. An antibody that has been shown to be suitable
titissue block. A surgical pathologist (to establish optimal per- for immunohistochemical staining procedures;
formance) has evaluated the performance of this test. Following ­optimally diluted with antibody diluent

594
 20
Fine-Needle Aspiration Biopsy Techniques

15. 3% sodium hydrogen peroxide structures immunoreactive for the antigen detected by each
16. Cytospin slides (Shandon) antibody used in the immunohistochemical stains selected to
17. Cytospin 3 (centrifuge for making cytospins evaluate the individual case. The immunoreactivity of the pri-
­Shandon) mary antibody and of the entire immunohistochemical staining
protocol is applied to a specific case.
Working Solutions
1. Dako target retrieval solution (1×) Procedure
a. 100 mL of Dako concentrated target retrieval solu- 1a. Cut paraffin sections at 3 μm and mount on charged
tion (10×) slides that have a control tissue section at the top in
b. 1000 mL of distilled water the “control” section of the slide. Label slide with case
Prepare fresh everyday number, block number, and stain.
1b. Prepare Cytospins (see previous description for
2. Dako target retrieval solution pH 9 (1×) ­cytospin preparation).
a. 100 mL of Dako concentrated target retrieval solu- 2a. Dry paraffin slides in 60ºC oven for minimum of 60
tion (10×) minutes.
b. 1000 mL of distilled water 2b. Cytospins, aspirates, and smears are fixed in anhy-
Prepare fresh everyday drous acetone for 15 minutes at 4ºC then air-dried
for a minimum of 35 minutes.
3. Wash buffer solution 3. Deparaffinize sections in 3 changes of Xylene—five
a. 100 mL of Dako wash buffer concentration (10×) minutes each.
b. 1000 mL of distilled water 4. Rehydrate section in decreasing grades of alcohol for
Expiration: 5 days 3 minutes each: 100% two changes, 95%, 80%, and
then rinse in distilled water.
4. 5% normal goat serum 5. Treat slides in 3% hydrogen peroxide for five minutes.
a. 5 mL of normal goat serum (Dako) 6. Paraffin section specimen pretreatment: Place slides
b. 95 mL of reconstituted Sigma buffer in heated target retrieval in steamer. For target
c. 2 drops of blue food coloring retrieval: 20 minutes; for pH 9: 30 minutes. Allow
Expiration: 1 month slides to cool on counter for 20 minutes, and then
they may be placed on the autostainer.
5. DAB chromogen solution (Dako K3466) 7. Block nonspecific protein binding sites by incubation
a. 1 drop of DAB chromogen for 5 minutes in normal goat serum.
b. 1ML of substrate buffer 8. Rinse for 2 minutes in Dako wash buffer.
Make fresh every day. 9. To the test slide add appropriate primary antibody
using the optimal dilution in antibody diluent, and
Controls to the negative control slide add the anti-mouse/anti-
Using one unstained slide from each case, the primary antibody rabbit immunoglobulins. Be sure that the section is
is replaced by a cocktail negative control antibody containing completely covered with solution for recommended
anti-mouse and anti-rabbit immunoglobulins. All other immu- incubation times.
nohistochemical-staining steps are carried out according to this 10. Rinse for 2 minutes in Dako wash buffer.
protocol. The “negative control” slide serves as a test for non- 11. Add HRP Envision polymer, incubate for 20–50
specific binding of immunoglobulins. Because most immuno­ minutes (incubation time varies, depending on the
histochemical stains are run in panels that include several primary antibody).
primary antibodies applied to several slides, the slides stained 12. Rinse in 2 changes of Dako wash buffer 2 minutes
for antibodies that do not react with the cells in question serve as each.
additional negative controls for the immunohistochemistry pro- 13. Place slides in DAB or DAB+ substrate solution for
cedure. For each primary antibody, histologic sections of various 5–8 minutes. Substrate should be prepared just
tumors and normal tissue known to contain cells positive for before use. (Incubation times and chromogens vary
the antigen detected by the primary antibody are stained for that depending on primary antibody.)
antigen. These positive control tissues serve as tests of the specifi- 14. Rinse for 2 minutes in Dako wash buffer.
city and immunoreactivity of the primary antibodies. 15. Counterstain slides in Gill’s III hematoxylin for
The tissue sections, when possible, are placed on the same 30 seconds.
slide as the patient tissue sample. In addition, patient sample 16. Blue slides in running tap water for 2–3 minutes.
sections stained for various antigens often contain normal ­tissue 17. Dehydrate, clear and cover slip.

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