2 sample preparatory techniques 2020

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JN CPII Sampling Techniques - 2020
Sample Preparation
Techniques
Introduction
 Quality of cytological diagnosis largely depends
on two aspects.
1. Excellence of clinical procedure used to secure
the sample (rubbish in, rubbish out).
2. Laboratory procedure used to process the
sample.
 At least ½ to ⅔ of false -ves are the result of patient
condition at time of sample collection and skill and
knowledge of individual collecting the specimen.
Principles and techniques of specimen collection
 Main concern to the Cytologist is whether the
specimen contains neoplastic cells.
 Characteristic of neoplastic cells is their tendency to
lose cohesiveness.
 Cells that lose their stickiness tend to exfoliate readily.
 If the neoplasm involves the surface of an organ then,
all what is needed is a device to sweep up the cells.
 The sweeping up process is the basis of exfoliative
cytology.
 For a neoplasm involving a body site that is out of
direct contact with a tissue surface it is unlikely to be
accessible using any of the methods of exfoliative
cytology.
 More sensible to use a needle and syringe to suck cells
from suspicious areas.
 This is the basis of aspiration cytology or, more
specifically, fine needle aspiration cytology (FNAC)
Principles and techniques of specimen collection
Exfoliative cytology
 Harvesting of exfoliated cells involve scraping,
brushing, or washing body surfaces or cavities.
 Simplest of all is the collection of body fluids into
which cells have spontaneously exfoliated.
 Typical examples are voided urine cytology and
sputum cytology.
Scraping, brushing, or washing from tissue surfaces
 Best known application of the scraping technique is
the cervical sample, because of its use in cervical
screening.
 Devices for collection have changed over time but
all are designed to sample the transformation
zone of the cervix.
 Area that most cervical neoplasms originate

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Sampling Devices
Notice that all these devices have one thing in common –
their shape matches the anatomical profile of the cervix
Preparation: patient’s instructions
 Schedule appointment 2
weeks after LMP.
 No tampon use, vaginal
medication or douches 48
hrs before appointment.
 No intercourse 24 hrs
before appointment.
 Explain exam.
Equipment
 Drape
 Exam table with
stirrups
 Exam light
 Speculum of proper
size
 Collection devices
 Supplies
Equipment
Thin-bladed Pederson speculums are most comfortable for most
women. For women who have had babies, the wider Grave’s speculum
may be needed for good visualization of the vagina and cervix
Supplies
Conventional Kit Thin Prep
Examination
 Wash hands
 Put on gloves
 Latex?
 2 pair

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Warm Speculum
 Warm water
 Not too hot
 Lubricates speculum
 Don’t use greasy material
such as vaseline to
lubricate speculum.
 Makes smears unreadable
on the microscope.
Gynecological Specimen Collection
 Principle
 The detection of cervical cancer and its precursors
as well as other gynaecologic abnormalities is the
primary purpose of obtaining a cervical cell sample.
 To this end, it is important to obtain a specimen that
is not obscured by blood, mucus, inflammatory
exudates or lubricant
Sample collection techniques
 Choice of method will depend on information to
be gained.
1. Aspiration from the posterior fornix.
2. Scraping of the cervix.
3. Scraping from the lateral vaginal wall.
4. Aspiration of the endocervical or endometrial
cavity.
1. Aspiration from the posterior fornix
 Instrument required: slightly bent glass pipette of
~0.5 cm bore & ~15 cm long.
 Method:
1. With bulb attached allow pipette to glide into the
posterior fornix.
2. Bulb must be compressed until the tip of the pipette
is in position.
3. Gradually release the compression to suck fluid
contents.
4. Spread material gently and quickly over the slide
and fix while wet.
Advantages of the method
1. Material may contain cells from the whole genital
tract increasing chances of lesion detection.
2. Good for hormonal studies because of the mixed
pool of cells.
3. Collection of material is easy, painless and need
not be performed by a medically qualified person.
Disadvantages of the method
1. Cell variety is large and hence the population of
each cell type is less.
2. Method is unreliable for detection of cervical
cancer in cases of uterine prolapse.
3. Age of cells in the aspirate is uncertain and older
exfoliated cells are usually in a poorer state of
preservation.

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2. Scrapping of the Cervix: Ayre’s spatula
Concave end to fit the
cervix.
Convex end for vaginal
wall and vaginal pool
scrapings
Ayre’s spatula
 Use concave end
 Rotate 360 degrees
 Rotate the spatula clockwise
beginning and ending at 9:00
or counterclockwise beginning
and ending at 3:00.
 Smear sample with a single
stroke motion using moderate
pressure to thin out clumps of
cellular and mucus material.
The correct way of depositing cells from an Ayre
spatula onto the glass slide
Advantages of the method
1. Produces an aggregate of cells derived from the
cervical surface only.
2. Since cells have not been spontaneously shed,
they wont show damage due to autolysis after
exfoliation.
3. Likely hood of detecting abnormal cells is greater.
Disadvantages of the method
1. Slides must be prepared by a trained person.
2. Area screened is limited to the reach of the
spatula.
3. Method is impractical for hormone studies
because cervical epithelium is less sensitive to
hormonal stimuli.
4. Wooden spatulas trap material which can lead to
false negatives.
 The endocervical brush should be introduced gently into
the canal and rotated with a gentle movement of 180
degrees.
 If the brush is inserted with force and with a harsh back-
and-forth movement, the following may occur:
 Smear obtained will be composed mainly of blood
 Endocervical cells will be distorted, showing marked pulling
artifact, and muscle fibres.
 To transfer material roll the bristles across the slide by
twirling the brush handle..
B. Scrapping of the Cervix: Cytobrush

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B. Scrapping of the Cervix: Cytobrush
 Push the cytobrush into
the canal, no deeper than
the length of the brush
(1.5 cm - 2.0 cm).
 Rotate only 180 degrees
(otherwise it will cause
bleeding).
JN CPII Sampling Techniques - 2020 JN CPII Sampling Techniques - 2020
Ads and disads of the method
 The pliable plastic bristles allow insertion of the
narrow tip into a small cervical os, e.g. in cases of
menopausal atrophy.
 The instrument is highly effective in obtaining
endocervical material and in collecting large
numbers of cells.
 One problem with the Cytobrush is the trauma that
occurs to the cervix as a result of the stiff bristles on
the instrument.
 To use the “broom,” long central bristles are inserted
into the os or until the lateral bristles bend against
the ectocervix.
 The instrument is rotated with gentle pressure
against the cervix a total of 3-5 times in clockwise
direction.
 To transfer material, smear sample with a painting
action, using both sides of the broom.
B. Scrapping of the Cervix: Cervex brush
Ads and disads of the method
 The Cervex Brush combines features of both the
spatula and the cytobrush and allows simultaneous
sampling of both the endo- and ectocervix.
 Major advantage: adequate amount of very well
preserved cytologic material is obtained from both
the ecto and endocervix.
 Most expensive of all the available sampling
devices.
C. Scraping of the lateral vaginal wall
 Instruments required: vaginal speculum, ordinary
wooden or plastic spatula.
 Spatula is introduced into the vagina under direct
vision through a speculum.
 Upper third of lateral wall is lightly scraped and
material is spread on slide and wet fixed.
Advantages & Disadvantages
 Recommended method for study of hormone
effects.
 May also be used for studying vaginal inflammatory
conditions.
 The technique has no place in cancer cytology.

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D. Aspiration from the endocervical
canal or endometrial cavity
 Instruments required: vaginal speculum, sterile
syringe with attached cannula or polyethelene tube.
 Method: Cervix is exposed with a speculum.
 Cannula or tube is then introduced into the
endocervical canal under sterile conditions.
 When in position fluid is aspirated by retracting the
plunger of the syringe.
 Treat material as for the other samples.
JN CPII
Endometrial aspirator
Sampling Techniques - 2020
Advantages & Disadvantages
 Lesions of the endocervix and endometrium are
detected with greater accuracy and ease.
 Skilled operator required to assure sterile
conditions of smear taking.
 Introduction of cannula into the uterine cavity may
be painful and is potentially dangerous.
Collection Devices
 Used for sampling endocervix, TZ and ectocervix.
 Endocervical brushes
 Wooden and plastic spatulas
 Plastic “broom-type” samplers
 Cytobrush and spatula together provide the best
specimen for cervical cytology (Boom et al., 1989).
 Choice of particular device will depend on size and
shape of the cervix and clinical situation.
Fixation
 Purpose: To maintain, as closely as possible, the
cytomorphologic characteristics and diagnostically
essential cytochemical elements of the cell.
 Functions of an appropriate fixative
1. Penetrate cells rapidly
2. Minimize cell shrinkage
3. Maintain morphologic integrity
4. Deactivate autolytic enzymes
Fixation
 Functions of an appropriate fixative
5. Replace cellular water
6. Facilitate diffusion of dyes across cell boundaries
7. Help cells adhere to a glass surface
8. Provide consistent results over time
9. Produce a permanent cell record
10.Stop cellular and microbial growth (anti-microbial)

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Fixation
 Historically, Papanicolaou used ether and 95% ethanol
(1:1) as fixative of choice.
 Currently used fixatives
 95% ethanol
 80% isopropanol
 100% methanol
 Carnoy’s fluid
 Schaudin’s fliud
 Various commercially available spray fixatives
Fixation Methods
 Wet Fixation
 The cell sample is immediately submerged into the
fixative solution.
 The cell sample remains in the fixative solution until
it arrives in the laboratory, where it is accessioned
and stained.
 The cells are not exposed to air at any time during
this fixation method.
Fixation Methods
 Wet Fixation with Air Drying
 Cell sample is immediately submerged into fixative
solution, then removed after a specified time
 The cell sample is then air dried and placed into a
container for transportation to the laboratory.
 In the laboratory, the slide is placed in 95% ethanol
or its equivalent prior to staining to rehydrate the
cell sample.
Fixation Methods
 Spray Fixation
 Immediate fixation of the wet cell sample with a
commercially available spray fixative.
 The spray-fixed cell sample is allowed to air dry and
then placed in a container for transportation to the
laboratory.
 In the laboratory, the polyethylene glycol (carbowax)
cell coating must be removed prior to staining.
Spray Fixation
 Spray fixatives usually consist of an alcohol base and
a waxy substance (carbowax) that provides a thin
protective coating for the cells.
 To remove the waxy coating, the slides are placed in
two separate rinses of 95% ethanol or two separate
rinses of tap water.
 Slides are placed in the first ethanol rinse for
approximately 30 min.
 95% ethanol is more effective and less time-consuming
than water rinses
MOST IMPORTANT: The most critical aspect of fixation once the specimen is
obtained, is to make the smear and then apply fixative IMMEDIATELY, making
sure that the entire surface is covered evenly with fixative material.

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Liquid Based Cytology
 Simply means cytology (study of cells) through a
liquid medium.
 Cells collected from the cervix (or any other site)
are placed directly into liquid preservative, than
transferred to a slide.
 avoids desiccation
 reduces the quantity of obscuring material.
Technique
 Liquid-based Pap test technology (SurePath® and
ThinPrep®) requires placement of cervical samples
into a vial of liquid preservative).
 During processing, non-diagnostic debris is partially
removed and the number of white and red blood cells
is markedly reduced.
 Fixed cells are then sedimented (SurePath) or filtered
(ThinPrep) as a thin layer on a slide and stained.
Advantages
 LBT results in a reduction in unsatisfactory slides
relative to conventionally prepared slides.
 Rapid fixation
 Even distribution of cells over a smaller slide area
 Decreased obscuring background elements, such
as blood, inflammation, and mucus.
Disadvantages
 More costly than conventional pap smears
 Preparation is more labour intensive
 Loss of background material
 Some differences in architecture and morphology
 Requires training for both the screeners and the
smear takers
Conventional versus Liquid based
 The conventional (Pap) test is considered
suboptimal due to false negative and false-positive
test results.
 This is caused by the poor quality of sampling and
preparation (obscuration by blood or inflammation,
bad cell fixation, and inhomogeneous distribution of
cells) and by errors in detection and interpretation.
 Liquid based cytology (LBC) was developed as an
alternative.
 Because only a representative portion of the sample
is used, the residual material in the vial may be used
for ancillary testing such as human papillomavirus
(HPV) testing and other molecular tests.

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Conventional Pap Smear
 Marjority of cells not
captured
 Non-representative
transfer of cells.
 Clumping and overlapping
of cells.
 Obscuring material.
 8000-12000 Sq. cells
Liquid-based Cytology
 Virtually all cells of sample
are collected.
 Randomised
representative transfer of
cells.
 Even distribution of cells
 Minimises obscuring
material
 5000 Sq. cells
LBC versus Conventional smears
Good cellularity: (obj. 5x) Sampling Techniques - 2020
JN CPII
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2 sample preparatory techniques 2020

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