Sampling and Fixation in

Cytology
Mr. Mohammed H Elhoweris, MSc, MIBMS
 Methods of Specimens collection
1- Exfoliative Cytology:
- It is the study of cells that have been shed or
removed from the epithelial surface of various
organs.
- Usually cells exfoliated during maturation,
infections, or Malignancy.
- Exfoliative Cytology is a simple, rapid, cost-
effective procedure for evaluation of cytology
samples
 Types of exfoliative cytology
1- Imprints:
- Touch imprints may be made directly from crusted
and ulcerative skin lesions or from impressions of
deeper surgical biopsies gently rolled onto a glass
slide prior to placement in fixative.
2- Scraping (Abrasive cytology):
- Collecting device such as spatula or brush are used
to gently scrap the lesion or epithelial surfaces, then
spread/buttered across a slide. (Oral cavity, Cervix,
Brochial , ….)
3- Squash Preparation:
- Biopsy or clot usually placed between 2 slides then
gently and smoothly drawn over the length of each slide.
4- Washings:
- Washings can be collected from various body sites.
Small amount of balanced saline solution are washed
over a directly visualized area and removed immediately
with suction.
- These washings are usually submitted unfixed to the
laboratory. If a delay is expected, they may be partially
fixed in 50% ethanol equal to the volume of the
specimen
- Usually used for respiratory tract and GIT
5- Discharge from nipple of the breast:
- Spontaneous nipple discharge and discharge produced
by breast massage are collected by applying the slide
directly to the nipple followed by immediate fixation.
6- Bronchoalveolar lavage (BAL)
- is a diagnostic procedure of washing a sample of cells
and secretions from the alveolar and bronchial airspaces.
- It is performed by installing commercial sterile 0.9%
saline solution for intravenous use via a channel of a
fiberoptic bronchoscope
- The fluid is then immediately withdrawn. The instilled
fluid fills the airspaces distal to the tip of bronchoscope,
replacing the air.
7- Body fluids:
- Serous or “body cavity fluids” are usually collected
with aseptic technique by needle puncture and
aspiration of the body cavity fluid
- Fluid samples are obtained from sampling body cavity
effusions (Pleural, Pericardial, Peritoneal), cysts , joints,
cerebrospinal fluid(CSF), and urine.
- Pleural, pericardial and peritoneal fluids can be
collected in tubes or syringes that may be either plain
or containing heparin or EDTA to prevent coagulation
- If immediate processing is not possible, it can be
preserved in the refrigerator for a period of 24-48 hours
2- Fine Needle Aspiration Cytology
• This is a technique used to obtain material from
organs that do not shed cells spontaneously (Solid
organs).
• It is valuable in diagnosis of lesions of the breast,
thyroid, lymph nodes, liver, lungs, skin, soft tissues
and bones.
• A fine or thin needle is defined as 22 or higher
gauge. (22-25 g)
• FNAB is a minimally invasive, cost-effective
technique with high diagnostic accuracy (in the
range of 90 to 99%)
A- Palpable Masses:
- It is useful in lesions that are easily palpable,
like growth of skin, subcutaneous soft tissue
tumours, thyroid, lymph nodes, salivary glands
and breast.
B- Non Palpable masses (Deep seated organs):
- Guided aspiration by internal imaging
techniques like C.T or ultrasonography allows
FNA of lesions of internal organs like lung,
mediastinum, abdominal and retroperitoneal
organs, prostate etc
 FNAC Procedure
1- Once the mass is stabilized between the
operator’s fingers, the fine gauge needle is
inserted into the mass.
2- When the needle is seated comfortably in the
mass, negative pressure is applied to the
plunger/syringe.
3- moving the needle back and forth within the
mass. This procedure should be repeated at
least 3 – 4 times at different angles within the
lesion to obtain a representative cell
population from the lesion in question
CT Scan guided FNAC
 Advantages of FNA
• Simple
• Quick
• Accurate
• Acceptable to the patient
• Multiple aspirations can be done
• Cost effective.
• Ancillary testing (Cell Block,
Immunocytochemistry, Molecular techniques).
Comparison between Tissue Biopsy and FNA
 Fixation in Cytology
• Fixation is the maintain of cell morphology
closely as possible to the living state, and
preservation of cytochemical elements.
• Rapid fixation of smears is necessary to
preserve cytologic details of cells spread on a
glass slide
 Properties of Cytologic Fixatives

● Penetrate Cells rapidly

●Do not excessively shrink or swell cells.
● Do not distort or dissolve cellular components.
● Inactivate enzymes and preserve nuclear details.
● Kill microbes.
● Improve optical differentiation and enhance
staining properties of the tissues and cell
components.
● Be matched with subsequent staining method
used.
 Wet Fixation
• The process of immersing of freshly prepared
smears immediately in a liquid fixative is called
wet fixation.
• This is the ideal method for fixing all
gynecological and non-gynecological smears
and any of the following alcohols can be used
1. 95% Ethyl Alcohol (Ethanol):
The ideal fixative recommended in most of the laboratories
for cytological specimen is 95% ethanol alone. It produces
the characteristic effect desired on nucleus. It is a
dehydrating agent and causes cell shrinkage as it replaces
water. But it causes only the desired amount of cell
contraction to yield optimal chromatin detail characteristics
of cytological preparations
2. Ether alcohol mixture:
- This fixative was originally recommended by Papanicolaou. It
consists of equal parts of ether and 95% ethyl alcohol. It is an
excellent fixative, but ether is not used in most of the
laboratories because of its safety hazards, odour and
hygroscopic nature
3- 80% Propanol and Isopropanol: Propanol and
Isopropanol cause slightly more cell shrinkage
than ether-ethanol or methanol. By using
lower percentage of these alcohols the
shrinkage is balanced by the swelling effect of
water on cells. Hence 80% propanol is a
substitute for 95% ethanol.
 Spray fixation
- Coating fixatives are substitutes for wet
fixatives. They are either aerosols applied by
spraying the cellular samples or a liquid base,
which is dropped onto the slide.
- They are composed of an alcohol base, which
fixes the cells and wax like substance, which
forms a thin protective coating over the cells
e.g. Carbowax (Polyethylene Glycol) fixative.
• The distance from which the slides are
sprayed with an aerosol fixative affects the
cytology details.10 to 12 inches (25-30 cm) is
the optimum distance recommended for
aerosol fixative
• Prior to staining, the slides have to be kept 10
mins in 95% alcohol for removal of the coating
fixative.
 Air drying fixation
• Air dried smears usually subsequently fixed
with alcohol
• Used with Romanowsky’s stains (Diff Quick,
MGG, Wright stain)
• Suitable for Non Gynecologic samples specially
lymph node, breast, and thyroid.
 Bloody samples
• The presence of RBCs that particularly or completely
obscure epithelial cells should be reported as either
less than optimal, or unsatisfactory.
• Usually fixative containing Glacial acetic acid are
used as a lysing fixative to treat bloody samples (eg;
Carnoys , Clarke’s )
• 2M Urea also used to remove RBCs from bloody
smears
• Prolonged fixation with GAA may affect cell
morphology (shrink, round up, … etc), so time for
pretreatment should not exceed 3 mins
Safety in Cytology Lab
• Special precautions are to be followed in all
laboratories using any body fluids, such as
sputum, effusions, and urine, because of the
potential to transmit disease-causing organisms.
1. Follow all instructions carefully.
2. Use gloves and goggles in all laboratory
experiments that involve the use of body fluids.
3. All contaminated material, such as slides,
coverslips, toothpicks, lancets, alcohol swabs,
etc., must be placed in a biohazard bag for
proper disposal and should never be reused.
A biosafety cabinet (BSC) — also called biological safety cabinet or
microbiological safety cabinet — is an enclosed, ventilated laboratory workspace
for safely working with materials contaminated with (or potentially contaminated
with) pathogens requiring a defined biosafety level
Risks associated with common hazards in the cytology laboratory

Hazard Risk

Electrical equipment Spark and fire

Glass and plastic Cuts and infection

Needle stick (FNA) Infection (HIV, HepB)

Unfixed cellular material Contact with eye or skin as result of

spillage or leakage

Alcohol , xylene Flammable

Giemsa stain Corrosive ,irritant

Papanicolaou stain Hydrochloric acid corrosive

George N Papanicolaou (1883 – 1962)