Glassware 1. Microslide coverslips 24 × 50 mm thickness 0.17 mm. 2. Measuring cylinder. 3. Coplin jar. 4. Staining jar. 5. Conical flask. May-Grünwald-Giemsa (MGG) Stain

 The usual internal diameter of the needle is 0.6 to

0.7 mm. When the needle reaches the intrathoracic mass,

the aspiration is performed as per palpable lesions. The

patient is asked to hold his breath during the aspiration.

The entire procedure of inserting the needle and aspirating the lesion should not take more than 20 seconds.

The needle may be reinserted in a different direction if

no representative material is obtained initially. After the

aspiration cytology, the patient needs to be kept under

observation for about 2 hours to detect rare complications

like bleeding or pneumothorax if any.

Liver

An 8–16 cm needle with external diameter of 0.6 mm

(22-gauge) are used for aspiration of liver (Usually, a

prothrombin time is estimated before liver aspiration. If

the value is very high, vitamin K is given to lower it to a

satisfactory level).

No local anesthesia is necessary. Depending upon the

site of lesion, the needle is introduced transcostally or

subcostally. The ideal transcostal area for entry of needle

is 9th intercostal space in midaxillary line. When the liver

is enlarged particularly when the tumor mass is palpable,

aspiration can be carried out in the subcostal region.

Prostatic: Aspiration is undertaken with the help of a needle

guide and long needle (20 cm). The patient is placed in

the lithotomy position. The prostate is carefully palpated

through rectum before the procedure. Well lubricated

gloved index finger of left hand with the needle guide is

fixed over the nodule palpable through rectum. Then

the needle is introduced through the needle guide and

aspiration is performed as described for transcutaneous

biopsy.

Ovarian tumor: Large ovarian tumors, which are palpable

per abdomen can be aspirated transabdominally avoiding

the loops of intestine. The lesion can also be approached

through the vaginal vault with the help of a needle guide

and a long needle as is described under the aspiration of

prostate.

Advantages of the Procedure

1. Fine-needle aspiration biopsy is a quick, convenient,

economic and almost painless procedure, which can

be practiced on an outpatient basis.

2. Local anesthesia is not required.

3. Can be attempted at multiple sites and repeated if

necessary.

4. Malignancy can be confirmed or excluded in potentially operable lesions suspicious of malignancy and

the extent of surgery can be planned well in advance.

5. Is a good diagnostic aid prior to application of radiation

in inoperable cases or where surgery is contraindicated.

6. By way of evacuation of a cyst content, it helps as a

therapeutic aid in addition to providing diagnosis.

7. It helps in assessing the stage of the disease prior to

surgery or radiotherapy.

8. Local recurrence or metastasis can be detected in

postoperative or post-radiation follow-up cases, for

further management.

9. Aspirated material can be used for immunological,

cytochemical, cytogenetical and microbiological

studies.

Limitations

False negative results may be obtained in the following

situations:

FIG. 26.1: Technique of fine-needle aspiration biopsy

A. Needle within the lymph-node

B. Retraction of the piston of the syringe in order to create negative

pressure in barrel of syringe

C. Movement of needle in 2 to 3 directions/plains

D. Release of piston and withdrawal of the needle

A

B

C

D

Cytology 811

1. If there is extensive fibrosis and sclerosis in a tumor.

2. If the tumor is highly vascular.

3. If there is tumor necrosis.

To minimize these errors, special precautions can be

taken. Wherever limitations exist, suggest an excision/

open biopsy.

Ultrasound-Guided Fine-Needle Aspiration Cytology

The use of ultrasound as a tool in medical diagnosis is

gaining increased acceptance in most medical centers. The

chief advantages of ultrasound as a diagnostic modality

are three:

1. It is a noninvasive study, causing little or no discomfort

to the patient and usually requiring no special preparation.

2. It does not require the use of ionizing radiation such as

X-rays. Studies to date have shown no proven adverse

effects from the ultrasonic beam at the conventional

power levels used for diagnosis.

3. Ultrasound is capable of providing some diagnostic

information, which may not be available using other

noninvasive techniques.

The ultrasound beam is in many ways similar to a beam

of light. It obeys the laws of optics and can (unlike X-rays)

be focussed, reflected, or refracted. The beam consists of

high frequency sound waves generated by vibration of

a piezoelectric crystal within an ultrasound transducer.

The crystal vibrates in response to an electrical signal,

the frequency of vibration being a function of the shape

and thickness of the crystal itself. This is exactly the same

principle that governs the sound of a bell. Bells of differing

shapes and sizes have different sounds. For most medical

applications, the frequency used is approximately 2.5

million cycles per second (2.5 mHz). The same crystal that

transmits the ultrasonic beam also functions as a listening

device. For example, a pulse of ultrasound is beamed for a

fraction of a second and the crystal then “listens” during

a much longer interval for the echo response. Returning

sound waves (echoes) strike the transducer, producing

vibrations which are transmitted as electrical signals to an

oscilloscope or for storage on the screen of a cathode ray

tube.

What produces these echoes? The tissues of the body

vary from each other in sound-transmission characteristics

(acoustic impedance). When two tissues of differing acoustic

impedance are apposed, the ultrasonic beam will be partially

reflected at the interface between them, returning an echo

signal to the transducer. The degree of difference in acoustic

impedance will determine the strength of the returning

echo. Thus, if soft tissue lies next to bone, which has a very

high acoustic impedance, or next to air, which has a very

low acoustic impedance, strong interfaces will be formed,

and strong echoes will be returned. On the other hand, soft

tissues (vessel walls, septa, fat, parenchyma, etc.) differ only

slightly from one another in acoustic impedance and the

echoes that are returned from their various interfaces are

relatively weak. These echoes are recorded as spikes on an

oscilloscope or stored as dots on the screen of the cathode

ray tube. This latter type of storage display is called a B-scan,

B because the brightness (and size) of the dots on the screen

varies with the strength of the acoustic interface.

B-scanning

B-scan displays are used when performing studies of the

abdomen, retroperitoneum, and pelvis. The orientation of

the dots on the storage screen varies with the orientation

of the transducer relative to the patient’s body. As the

transducer (attached to a rigid hinged arm, which holds it in

any plane selected) is moved across a section of the patient’s

body, it sends a narrow, well-directed ultrasonic beam

through the tissues. As this beam traverses the abdomen, it

is partially reflected at various interfaces, owing to relative

differences in acoustic impedance. These reflected echoes

are recorded as dots, which build up an image of the section

on a storage screen (Fig. 26.2). When a suitable picture has

been made, it may be photographed on either Polaroid or

X-ray film, or recorded on heat-sensitive paper.

It is important to remember that relatively inhomogeneous tissues, such as solid organs or masses, will

generally have many weak echoes recorded within them,

representing small vessels, ducts, and septae traversing the

tissue. Relatively homogeneous tissues, such as fluid-filled

organs or cystic lesions, show a few internal echoes, even

FIG. 26.2: As the transducer is moved across the body, an image of the

body section traversed is built up on the storage screen

812 Concise Book of Medical Laboratory Technology: Methods and Interpretations when the sensitivity of the machine is turned up very high

(“high gain”).

Ultrasonography-guided fine-needle aspiration cytology

is being practiced in case of lesions of thyroid, and some

selected cases of intrathoracic tumors and also in breast.

Advantages

1. Takes less time than to perform mammography.

2. No radiation hazards.

3. Can be used to guide the aspiration of cyst that could

not be drained clinically hence avoids surgery.

4. It can guide placement of wire guide.

Disadvantages

1. Limitations in diagnostic usefulness because benign

appearance overlaps, the malignant.

2. Unable to detect lesions less than 1.5 cm in diameter

(breast).

Application

Differentiating cyst from solid mass.

SMEARING TECHNIQUES

One-step Smearing Technique

The standard overlap and pull-apart method of smear

preparation can itself be a source of dilution. Aspirates of

poorly vascularized tumors often yield semisolid, undiluted

tissue fragments that can be easily expelled from the needle

core as a compact drop onto a slide. If standard pull-apart

smears are prepared, this material is dispersed onto two

slides. A slight modification of the pull-apart technique,

called the one-step technique, greatly limits what is in effect

a 50% dilution of the material. The two slides are held at

right angles. A small drop of aspirate material is placed near

the frosted edge of a slide held by one hand at that edge.

A second slide is then placed across the first, establishing

a fulcrum such that the mobile slide, when carefully

rotated away from the observer and downward toward the

material, just covers the drop. The smearing slide is then

quickly but smoothly brought toward the operator, with a

smearing pressure barely greater than that caused by the

capillary space dispersion of fluid between the slides. This

gentle and smooth stroke yields optimal monolayer smears

for semisolid or small-volume specimens. Little material is

transferred to smearing slide.

Two-step Particle Concentration and

Smearing Technique

For needle aspirates that are diluted by fluid, a procedure

called the two-step smearing technique provides an optimal

concentration of cells on the slide.

With the frosted ends away from the observer, each slide

is held at the tips of its lateral border by three fingers. The

thumb and index finger contact the upper outer frosted

corner of each slide, and the fifth finger contacts the

underside of the lower outer corner.

First, a drop of the aspirate is placed near the frosted

end of a clean, grease-free slide. With the labeled ends

still away from the observer, the other hand brings the

smearing slide onto the first slide, at a point between the

drop of aspiration and the observer. The smearing slide is

then passed away from the observer and through the drop

so as to collect the drop in the acute angle formed by the

two slides.

With the first slide still stationary, the mobile slide

begins the concentration procedure by next moving

the entrapped material away from the frosted end and

toward the observer. This movement disperses much of

the fluid over the central portion of the stationary slide,

while most of the particles remain with the small amount

of residual fluid at the line of slide intersection. At this

point, the mobile slide is lifted perpendicularly upward and

away from the stationary slide, and the similarities of this

technique to preparing peripheral blood smears end. Next,

the stationary slide is rotated vertically with its frosted end

pointing downward. For 1 to 3 seconds, gravity is allowed

to draw any excess fluid further away from the particles,

which largely remain at the last line of slide intersection. The

particles can frequently be seen by reflected light as small,

slightly raised points.

At this point the basic concentration maneuvres (first

step of the two-step technique) are complete, and the

particles now await monolayer smearing by a slight

variation of the standard pull-apart technique (the second

step). By fulcrum action, the mobile slide is placed so that

when slowly rotated onto the other slide it extends about

1 to 2 mm past the line of concentrated particles. As soon

as the two slides come into contact, they are gently pulled

apart with minimal additional pressure, yielding a wellformed monolayer (Fig. 26.3).

REQUIREMENTS FOR LABORATORY SET UP

Equipment

1. Handle constructed for disposable 20 mL or 10 mL

syringe (Cameco, Enebyberg, Sweden).

2. 10 mL or 20 mL disposable plastic syringe (ASIK,

Aps. Denmark).

3. Disposable needle of various sizes

 23G ×1” (0.6 × 25 mm)

 22G × 2” (0.7 × 50 mm)

 20G short and long.

4. Needle guide for transrectal biopsy (KIFA, Solna,

Sweden).

Cytology 813

5. Examination table.

6. Binocular microscope.

7. Centrifuge.

8. Analytical balance.

9. Cytocentrifuge.

10. Distillation plant.

11. Refrigerator.

12. Slide cabinet.

Glassware

1. Microslide coverslips 24 × 50 mm thickness 0.17 mm.

2. Measuring cylinder.

3. Coplin jar.

4. Staining jar.

5. Conical flask.

May-Grünwald-Giemsa (MGG) Stain

Reagents

1. May-Grünwald stain

2. Giemsa’s stain

3. Methanol

4. Glycerol

5. Conical flask

6. Phosphate (pH 6.8).