CYTOPREPARATOR

Y TECHNIQUES
AMA AFRAH

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 HISTORY
• In nineteenth century when John Lister developed a
microscope that was free of spherical and
chromatographic aberration, physiologist Schleiden and
Schwann were promoting the concept of cell being the
basic unit of life.

• These developments was to create a climate of exploration in science

and medicine that led to study of tissue fragments and body
secretions in light microscopy with the view of understanding
pathologic basis of diseases.

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 HISTORY
• Drawings of
• Henceforth there were scientific microscopic structures
contributions to cytological
studies worldwide; Johanns Muller
from Germany (1801-1858), Alfred
Donne French lawyer with interest
in microscopy (1801-1878),
Emeritus Prof of medicine,
King’s college S. Beale.
• These scientists published
articles on microscopic
appearance of ascitic fluid and
urine cytology using drawings
which correspond to current
concepts.
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 HISTORY
• One of the first Americans with interest in cytology was Dr. F.
Donaldson ( 1853) made impressions smears from cut surfaces of
tumours.
• French pathologist Henri Lebert aspirated cells from cystic and
solid
tumours.
• Gastric cytology we attribute to Rosenbach in 1882.
• Cytology of effusions and CSFs, Bennet in 1849.

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 HISTORY
• There was confusion about origin of cancer cells because the
mechanism of cell division and replication was not understood then.

• But Johann’s Muller brought some form of order into the chaos by
publishing a monograph on ‘ THE NATURE AND
STRUCTURAL CHARACTERISTICS OF CANCER
AND THOSE MORBID GROWTHS THAT MAY BE
CONFOUNDED WITH IT’.

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 HISTORY
• John Muller: • Benign /malignant cells
• The book described microscopic criteria
that distinguish benign from malignant
tumours and a lot of illuminating
statements that are true today.

• Physiologically all malignant tumours

were closely related ; demonstrating that
malignant cells exfoliated very easily than
normal cells.
• Classified tumours into 2 sarcomas and
carcinomas ; connective tissues
• All medical literature published then was
looking at how the study of cells could be
applied to clinical diagnosis.

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 HISTORY
• Exfoliative cytology
• Julius Vogel (1843) is credited with what is known today as exfoliative
cytology.
• Professor Baele (1860); sputum cytology

• Dr. Lambl (1856) has credit for urine cytology ; bedside test

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 HISTORY

• Unfortunately centuries later, the enthusiasm for exfoliative

cytology was not sustained.
• WHY?
• Histological techniques had perfected and diagnosis skills improved,
tumour typing in exfoliative cytology was not accurate.
• Presence of inflammatory cells in sample made it unsuitable for
cytological analysis.
• The speed with which one could get cytological report for quick
medical intervention was replaced by frozen sections in histology.

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 HISTORY
• Many years passed before exfoliative cytology was accepted as
valid method of scientific investigation because of persistent
advocacy.

• This credit we give to Dr. G. Papanicoulaou who through teaching

and research convinced his medical colleagues of the clinical value
of cytology with such a success that the technique is now used in
clinics and hospital throughout the world.

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 HISTORY
• Confirmation of some of his findings by other scientists led the
American cancer society taking up the challenge of cervical screening
by accepting the fact that vaginal smear was unique for detecting
uterine cancer.

• Henceforth advancement in cytology practice before and after

Papnicoulaou’s death proved that cervical scrapings were more
appropriate samples in diagnosing uterine cancers than vaginal.

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 HISTORY
• It was evident that the scope of our
cytology was not limited to tum s
cells but also hormonal changes ather
well as diagnosis of viruses and o
specific infectious agents.

• The most remarkable change in

cytology since the death of Dr.
G. Papanicoulaou is resurgence
of interest in FNA as a method of
cytological examination.

• Today cytological findings are

clearly related to underlying
histology.
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 GENERAL OVERVIEW OF
CYTOLOGY
• What is cytology? Audience

• Essence of cytology: screening, diagnosis

• Diagnostic tool : quick, safe, cost effective, simple, fast way
of
diagnosing without resorting to surgery.
• Screening tool: non-invasive tool for screening apparently
healthy/asymptomatic individuals for pre-cancer lesions.
• Hormonal change
• Diagnosis of viruses and other infectious agents. 12
 A LOOK INTO THE FUTURE
• PIT FALLS
• Despite important advances in cervical cancer screening there is
an estimation of 7-17% false negatives.
• WAY FORWARD
• Methods of sample collection should be improved.
• Efficient quality control.
• Introduction of universally structured training programmes which
are
lacking in many countries.
• New staining methods and development of antibodies. 13
 SITES OF SPECIMEN

• 1. Organs : bladder, lung, stomach, cervix, breast, skin,

mouth, muscle, thyroid, etc

• 2. Body cavities : peritoneum, pericardium, pleura etc

• 3. Body compartments: joints, etc

• 4. Palpable lumps: breast, muscle, lymph nodes, etc

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 BODY CAVITY
• Anatomic spaces in the body • Lined by serous membranes or
contains organs. mucosa
• Serous membranes made up of
mesothelial cells. Eg pleura,
peritoneum, pericardium
• Mucosa is made in part by
epithelium eg. Ependymal cells

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 BODY CAVITY
• Cells lining the cavities forms glands and produces serous secretions
and/or mucus
• Serous gland

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BODY CAVITY
• The pericardial cavity is lined by
inner visceral and outer parietal
serous membranes that produce
pericardial fluid that help the
heart in performing its duty of
receiving and pumping blood by
reducing friction.
• The purpose of the body fluids
are to help the organs perform
the functions without friction

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 ACCUMULATION OF FLUID IN
SEROUS CAVITIES

•.
• Under normal circumstance there is equilibrium between formation
and reabsorption of serous fluid

• Infected, infarction, malignancy or any disease condition, formation

of serous fluid exceeds reabsorption, therefore is accumulation of
fluid (effusion) in the cavity making it distended and may cause pain.

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 IMAGING DIAGNOSIS OF
EFFUSION
• C. T scan

• MRI

• X Ray

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 SAMPLE TAKING
• Body cavities are
punctured with needle
and fluid tapped

• CAUTION
• The procedure for
tapping samples from
these sites is invasive
so they must be handled
with cure.
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 TYPE OF SAMPLES
• EXFOLIATIVE • ASPIRATION
• Voided urine/bladder washing
• Sputum/bronchial lavage • FNA
• Gastric lavage • Pericardial fluid
• CSF • Peritoneal fluid
• Pap smear • Pleural fluid
• Buccal smear • Synovial fluid
• Semen • Etc
•
• Etc • Samples taken is
• Samples are voided or taken invasive
through mechanical
exfoliation.

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SAMPLE CONTAINERS FOR
FLUID SPECIMEN
• Clean, transparent, wide mouthed containers with a lid that can be
screwed

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APPEARANCES OF
FLUID SAMPLES

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 RECEIPT AND IDENTIFICATION
OF SPECIMEN
• Integrity of specimen must be checked; specimen should be accepted
only if the test is requested by a physician or a person authorized to
do so.

• Sample should be accompanied by request completed by the

authorized provider and signed.

• Specimen must be labelled ; name, age, gender, type of

specimen.
• Specimen Fresh or appropriately preserved.
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 REQUISITION REQUIREMENT
• Patient first and last names
• Age / date of birth
• Date sample was collected
• Site of specimen and name of specimen
• Diagnosis
• Clinical findings such as infection
• History of cancer/Chemotherapy/
• Name/phone number of requesting physician
• Any unique identifier
• Clinical history: LMP/Pregnancy/PM/hormonal therapy/IUCD/IUD/
previous abnormal cervical cytology / hysterectomy. etc

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 ACCESSIONING
• The information on sample and request card should be matched.
• Variation in information should be verified
• Any rectification must be documented.

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 CRITERIA FOR REJECTION
• There should be written criteria for rejection of specimen if
• 1. There is no name
• 2. No age,
• 3. Site of specimen
• 4. Requesting physician's name
• 5. When vital information is missing
• 6. Mismatch of information

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 PLANNING TO HELP THE
SITUATION
• Distance : how far away I’m I from Korle-bu or any facility that have it.

• Preservation
• Serous fluids contain protein that can sustain the cells to about 4
hours without refrigeration (note not freezing).
• If one cannot get to cytology unit within 4 hours, the transporter of
serous fluid must put sample on ice.
• Urine and any other fluid sample with the exception of CSF must be put
on ice even if , transporter will get the cytology unit that fast.

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PROCESSING OF CYTOLOGY
SAMPLE
• Macroscopic description of
specimen
• Record at the back of the request
card
• 1. Colour
• 2. Volume
• 3. Consistency/clarity
• 4. Name of person processing the
sample , date of processing,
number of slides prepared.

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PROCESSING OF FLUID
SAMPLES
• Clear sample means few cells and cloudy sample means many cells.

• Based on clarity of the fluid sample, you choose your method of

sedimentation.

• For very cloudy fluid, leaving the sample on the bench can separate
the cells from the supernatant by gravity.

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PROCESSING OF FLUID
SAMPLES
• The clear fluid, choose a speed
(3500 rpm) for about 3mins.

• For hazy fluid , choose moderate

speed (2500 rpm) for about
4min.
• Choose low speed (1500rpm)
for cloudy sample.

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 PROCESSING OF CYTOLOGY
SAMPLE
• After centrifugation, the cells are separated from the supernatant by
pipetting or decanting into disinfectant.

• Cell deposit is mixed using Pasteur pipette .

• Monolayer smears are prepared from the cell deposit.

• How do ensure your smear is monolayer?

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 SMEAR PREPARATION
• Squash
preparation / crash
and pull for
cloudy samples
• Put a drop of cell deposit on a
clean labelled slide.
• Put a second labelled slide
facedown on the first slide.
• Gently slide/pull apart the 2
slides with very little pressure to
prevent damage of the cells
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 TYPES OF FIXATION IN
CYTOLOGY
• 1. Wet fixation: Immediate immersion of smear in 95% ethanol for
at least 15 minutes

• Dry fixation :Smears are put on the bench to air dry and subsequently
put in absolute methanol for at least 15 minutes.

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 FIXATION OF SMEARS

• For cell that falls under exfoliative do only wet fixation.

• For cell that falls under aspiration both wet and dry fixation is done.

• Refer to slide 20
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 SMEAR FIXATION
NOTE
Cell that fall under aspiration, for
any 2 smears prepared at a time
through squash method, one is
fixed wet and the other dry so
that what ever is in the dry fixed
smear will be replicated in the wet
fixed as well.

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 ROUTINE STAINING IN
CYTOLOGY
• 1.Papanicoulaou staining technique

• Cocktail of Rowmanosky stain: Jenner-Giemsa, Wright-Giemsa,

Leishman- Giemsa, Toluidine blue

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 PAPANICOULAOU STAINING
TECHNIQUE
• Polychromatic staining technique that have an advantage
demonstrate
exfoliative cells best by ;
• 1.Give nuclear details
• 2. Cytoplasmic differentiation of squamous cells
• 3. Cytoplasmic transparency
• With 4 Main stages
• 1. Fixation in 95% ethanol
• 2. Nuclei staining in haematoxylin
• 3. Cytoplasmic staining with Orange ‘G’/ Eosin Azzure
• 4. Clearing in xylene and mounting a cover glass
• There is ethanol in between any 2 stains.
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 PAPANICOULAOU STAINED
CELLS
• Normal squamous/squamous carcinoma/mesothelial
cells/adenocarcinoma

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 LEISHMAN – GIEMSA
STAINING
TECHNIQUE
• It is a cocktail of Leishman and Giemsa stain.
• Demonstrate dried fixed smears best especially those from the
Haemic regions; blood related malignancies are demonstrated
better.
• Stain with Giemsa for 3-5minutes
• Flood with Leishman for 3 minutes
• Dry smears and mount

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ROWMANOSKY STAINED
CELLS
• Lymphocyte/lymphoma/thyroid carcinoma

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COMPARARISON OF LYMPHOMA
IN PAP STAIN AND
ROWMANOSKY

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