CYTOPREPARATORY

TECHNIQUE
AMA AFRAH
 CYTOPREPARATION
• Samples: gynaecological and non-gynaecological samples
• Gynaecological : Pap smear

• Non-gynaecological samples: any other apart from Pap smear;

• 1.Body fluids: urine, sputum, serous fluids( 3 Ps), synovial fluid, CSF, Semen,
amniotic fluid etc, bronchial wash/lavage, gastric lavage etc
• FNA: palpable and non-palpable lumps
GYNAE VERSES NON-GYNAE SPECIMEN
 CYTOPREPARATION
• Cytopreparation is the science of controlling cytomorphology for diagnostic
applications.

• It encompasses all those materials and methods that interact with cells from
specimen collection through cytologic preparation’s to microscopy

• Materials: stains, fixatives, equipment and devices etc

• Methods: specimen collection and specimen reception, processing, staining,
microscopy, filling(specimen, request cards, duplicate reports, reported slides
etc)
 CYTOPREPARATION
• Attention be paid to cytopreparation at all levels — academic, professional,
and regulatory
• Benefits of paying attention to can include cytopreparation includes
• 1. Optimized and standardized preparations

• 2. Documentation (filing) that exceeds regulatory requirements

• 3.Reproducibility: An institutional memory that helps ensure consistency

of outcome as personnel come and go; distribution of cells, and their final
size and shape, thickness, chemical composition, chromatin distribution
pattern, color, transparency (monolayering) and visibility
•
 CYTOPREPARATION
• 4. Control of process

• 5. Cost savings in time and materials

• 7. Tighter diagnostic classifications

• 8. Enhanced teaching capabilities

 CYTOPREPARATION

• 9. More instructive photomicrographs:

•
• 19. Reduced legal and fiscal exposure:

• 20. Enhanced professional pride.

• A technically well-run laboratory is not only good medicine, but also

good business.
 QUALITY CONTROL
• Quality control activities look forward; end product

• They define the product’s quality, imparting to it the credibility needed for its
intended purpose.

• QC activities are the result of careful planning and are applied

prospectively to everything that contributes to the final product, thereby
impacting the outcome.

• QC activities are deterministic (i.e., lead to expected results when followed).

 QUALITY ASSURANCE
• Quality assurance activities look backward.

• They measure the degree to which desired outcomes are successful

(i.e., their impact).

• QA activities, therefore, retrospective outcomes.

• The findings modify the processes that contribute to the final product
(e.g., did the patient have cancer as reported; if not, why?).
• As a practical matter quality assurance activities are probabilistic (i.e.,
have attendant uncertainty relative to reliability), as it not possible to
review all product outcomes
 QUALITY MANAGEMENT.

• QC and QA activities must be practiced continuously to monitor and

maintain the performance of the two sets of contributory processes

• 1. Recognize problems as they arise,

• 2. Identify corrective actions to be taken, and improve quality.

• Taken together (QC and QA), these two sets of activities constitute a
program of total quality management.
 ANALYZING QUALITY CONTROL AND
QUALITY ASSURANCE ACTIVITIES
• Personnel Proficiency: relates to the training, education, and
professional performance of the laboratory personnel.

• Cytopreparation: Successfully detecting abnormal cells is the outcome

of a series of interdependent samplings of successively diminishing size.

• The specimen collection technique samples the biologic process, the

cytopreparatory technique samples the specimen, the screening process
samples the preparation, and the diagnostic interpretation samples the
cellular features.
 QUALITY MANAGEMENT. QUALITY MANAGEMENT.
QUALITY MANAGEMENT

• High sensitivity :Optimized processes increase the probability of

abnormal cell detection

• Reduce false negatives: Reduce the incidence of missed

abnormal cells

• See clearly health status of all cells

 CYTOPREPARATION
• There is diversity of body sites of samples and biologic processes but all non-
gynecological cytologic specimens requires a simplifying, unifying approach to
quality control (QC) and quality assurance (QA) to ensure one obtain cells of
diagnostic significance.
• Procedures in cytopreparation
• 1. Sample/specimen collection: exfoliation, abrasion, brusing, aspiration (eg
FNA)
• 2. Specimen/sample reception
• 3. Sample or specimen processing: macroscopy, centrifugation, smear
preparation, appropriate fixation, staining
• 4. Microscopy
• 5. Filling
 CYTOPREPARATION: SAMPLING

• Sample collection: lavages balanced electrolyte solutions not saline otherwise cells
will lyse (ions are not balanced).

• Lesion is on epithelial surface: exfoliation, brushing, abrasion eg Pap smear, buccal

smear, skin etc
• Palpable lumps on skin: FNA

• Non-palpable lump within the body: image(X-ray, scan, MRI etc) guarded FNA

• NB. Unsatisfactory smear of FNA: check specimen collection technique or

preparation technique
 CYTOPREPARATION-SPECIMEN RECEPTION

• Accept fresh specimen or specimen in appropriate fixative

• Appropriate fixation: smears from epithelial surfaces(exfoliated, brushing, abrasion,)

should be wet fixed with………………?

• Fluid specimen: lavages, serous fluids, synovial fluid, CSF

• Body fluids: urine, sputum

• Specimen must be accompanied by appropriately filled request card with right

information
• Match information on request card and sample, if they tally accept and log into
records book and give pathology number-alpha numberic (GC/nth number/yr ;
NGC/nth/yr)
 CYTOPREPARATION-SPECIMEN RECEPTION

• Macroscopy

• Fluid specimen: Record volume , colour and consistency

• FNA: describe the nature of the sample aspirated

• Based on consistency (hypercellular, hypocellular) choose method of

centrifugation
FLUID SAMPLE WITH DIFFERENT CONSISTENCY
 CYTOPREPARATION-PROCESSING
• Fluid specimen and body fluid can be

• 1. Hypocellular specimen (clear fluid specimen): centrifuge the specimen to

concentrate the cells.
• Methods of centrifugation: conventional centrifuge, cytocentrifuge/cytospin,
membrane filtration etc

• 2. Hypercellular specimen (thick or cloudy): dilute sample as needed to avoid

overpopulating the preparation with electrolyte balanced solution not saline.

• 3. Mucoid specimen: sputum can be lysed using sputulysin

 CYTOPREPARATION-PROCESSING
• Preservation of fluid specimen

• Fresh specimen: protein in serous fluids can sustain viability of the cells for
up to 1 to 4 hour, beyond this time specimen should be put in fridge at 4
degrees Celsius or on ice during transportation to the lab

• If the cells are poorly preserved in a fresh specimen, they may have
degenerated because too much time has passed since collection, or the
specimen was suspended in normal saline, or storage conditions have
been substandard.
 CYTOPREPARATION-PROCESSING

• If the cells are poorly preserved in a specimen collected in

preservative, the preservative itself may have caused the cells to
swell and burst (e.g, certain dilutions of alcohol).
 CYTOPREPARATION-PROCESSING

• If particulates are observed, an attempt should be made to redissolve them if

possible (e.g., slightly acidifying fresh urine specimens with acetic acid to
redissolve phosphate salts) or to separate them by differential sedimentation.

• Particulates can be problematical for specimens that are processed

automatically in a monolayer device.
• The particulates can clog the filter pores and cause cell collection to stop
prematurely.
• Prepare cell block from particulate or tissue fragment.
 CYTOPREPARATION-PROCESSING
• Bloody fluid specimen should be collected into a heparnized tube
•
• Pick cells for preparation of smear from buficoat

• If RBCs outnumber nucleated cells (mesothelial cells for serous,

transitional/urothelial for urine ) excessively, they must be removed prior
to preparation to prevent their crowding out the nucleated cells and result
in a false negative.

• Bloody fresh specimens may be treated with saponin to haemolyse the red
blood cells.
 CYTOPREPARATION-PROCESSING

• Bloody preserved specimens may be suspended in one of at least two

proprietary hemolytic preservatives.

• Hemolysing the RBCs by immersion in a Carnoy-like fixative after the

cell spread has been made will not fix the problem

• Lysing fixatives for fixing bloody specimen

• 3% acetic acid in 95% ethanol for wet fixation

• 5% acetic acid in methanol for dry fixation

• Do cell block for blood clots
 CYTOPREPARATION-PROCESSING

• If tissue fragments are observed in fluid specimen, prepare cell block and
process just as in histology.

• Clear urine specimens, for example, appear to be acellular but in fact often
contain hundreds if not thousands of cells.

• Such specimens must first be concentrated by conventional centrifugation,

then examined microscopically to determine the appropriate cell
concentration method (e.g., membrane filtration,
cytocentrifugation/cytospin monolayer device) and how much specimen to
use.
 CYTOPREPARATION-PROCESSING

• Turbid specimens may appear to be hypercellular to the unaided eye,

when in fact the turbidity is due to light-scattering particulates and not
cells. Preprocessing to rid the particulates is indicated.

• So-called watery cytologic specimens such as urine and CSF are often
spread on albuminized slides, frosted slides, or albuminized-frosted
slides — all in an effort to keep cells from falling off slide when immersed
in alcohol.
 CYTOPREPARATION-PROCESSING
• Smear preparation

• Whether using raw cell suspension or resuspended cell concentrate,

transfer a small amount of cell suspension to the slide.

• Use less than you might think is needed. Only a single/mono layer (“a light
dusting”) of well distributed cells is required, not a heavy layer.

• To prepare mono layer, the specimen should not be able to flow to the
edges of the slide when a second slide is applied to spread the cells by the
2-slide pull technique.
 CYTOPREPARATION-PROCESSING

• Smear preparation

• You have succeeded when the slides resist sliding.

• If the cell suspension is expressed beyond the slide boundaries of slide, too
much sample was taken to prepare smear.
 CYTOPREPARATION-PROCESSING

• Fixation

• Wet fixation: Fix cells from epithelial surface/aspiration/ wet

(immediate immersion of wet smear in 95% for at least 15 minutes

• Dry fixation: aspirations ; air dry smear, fix in absolute methanol for
at least 15 minutes
 CYTOPREPARATION-STAINING
• Routine cytology stains are Pap stain and cocktail of Rowmanoski stain

• Papanicoulaou (Pap) staining technique: stain all wet fixed smear with
Pap stain

• Pap stain: haematoxylin, Orange Green 6 (Orange G 6), Eosin

Azzure/eosin alcohol (EA).

• Cocktail of Rowmanoski stains: stain all dry fixed smears with Cocktail
Rowmanoski stain
 CYTOPREPARATION-STAINING

• Performance Expectations

• Haematoxylin. All chemically competent haematoxylin formulations

produce satisfactory results when used properly.
• Staining times of 1 to 2 minutes are typical for Gill’s hematoxylins No. 1 and
Gill’s haematoxylins 2. Mayer’s Haematoxylin
• After bluing, A blue color = satisfactory; violet= insufficient bluing; gray =
borderline, brown = unsatisfactory.

• Chromatin particles visible in intermediate squamous cell nuclei and

chromatin particles in the lobes of well flattened neutrophils should also be
visible.
 CYTOPREPARATION-STAINING
• Haematoxylin
• Slight coloration of the cytoplasm after staining with haematoxylin is
permissible.

• Heavy coloration indicates the staining time is too long or the solution is
too concentrated ; Either decrease the staining time or dilute the solution.
 CYTOPREPARATION-STAINING
• Be certain to blue the stained slides sufficiently.
• Bluing can occur over a wide range of pHs.

• Low pHs (e.g., 5-6, distilled water), blue slowly over several minutes.

• Moderate pHs (e.g., 8-8.5 for Scott’s tap water substitute, tap water etc ) blue single
cells and thick tissue fragments and cellular clumps satisfactorily within 2 minutes.

• High pHs (e.g., 10-11, 1.5% NH4OH in 70% alcohol) blue rapidly within seconds and
tend to detach cells.
• Control bluing microscopically
 CYTOPREPARATION-STAINING
• Orange G and Eosin Azzure.
• Orange G should appear yellow to orange; 15 sec to 1 minute is the usual range
of staining times.
• EA is often problematic because of fundamental limitations in its chemical
composition. Ideally, one should see clearcut hues of green and red/pink in
separate cells.
• Staining times less than about 3 minutes usually favor the uptake of eosin Y,
with eosin and light green often occupying different areas of the same cells.

• Most EA formulations perform optimally in the 6-8 minute range.

• Note particularly that the OG and EA staining times are interdependent:
relatively too much time in OG will overload cells with orange G and block the
subsequent uptake of eosin.
 CYTOPREPARATION-STAINING
• Orange G and Eosin Azzure.
• Buccal Smears for Quality Assurance and Test Probes
• Alcohol wet-fixed buccal smears are invaluable probes
to determine the performance of each new lot of stain,
to select suitable staining times, to find out how many
slides can be stained satisfactorily by a given volume of
each stain, to learn when rinses should be changed, and
to troubleshoot whether a given stain already in use is
the cause of an observed staining problem.
CYTOPREPARATION-STAINING

• Orange G and Eosin Azzure.

• Buccal Smears for Quality Assurance and Test Probes

• However, they should be used when new containers of the same

stain with different lot numbers are opened to confirm that the stain
does indeed perform as expected.

• Manufacturers occasionally make bad batches of stain.

 CYTOPREPARATION-STAINING
• A series of buccal smears should be used to answer three questions for each
of the three staining solutions of the Pap stain:

• (1) what color is produced

• (2) what staining time is required to produce the desired optical density,
• (3) what is the distribution vis-à-vis the chromatin and cytoplasm.

• To confirm that each new batch of stain is performing as expected, stain

different sets of alcoholic wet-fixed buccal smears in haematoxylin for 30
sec, 1, 2, and 4 min; OG, 15 and 30 sec, 1 or 2 min; EA, 1, 2, 4, 6, and 8 min.
• Choose the staining time that appears best (see the immediately preceding
descriptions). Usually the times are close to those recommended.
 CYTOPREPARATION-STAINING
• Rinse the haematoxylin stained slides in two changes of tap water (10 dips each),

• blue per lab convention,

• rinse in two changes of tap water (10 dips each),

• dehydrate, clear, and mount.

• Rinse the OG and EA stained sets of buccal smears in 95% alcohol, dehydrate in
absolute alcohol, clear, and mount.
 CYTOPREPARATION-STAINING

• Orange G looks yellow in thin areas and orange (keratin) in thicker ones.
•
• Light green distinctly green in properly formulated stains

• Eosin is red in properly formulated stains. Both OG and EA have

phosphotungstic acid as mordant

• If the phosphotungstic acid concentration is too low, there may be little

or no differential staining—the green and red colors will be muddy and
dull.
 CYTOPREPARATION-STAINING
• Stain Duty Cycles: Duty cycle is the number of slides that can be stained
per unit volume of stain before a decline in quality is detectable.

• Buccal smears can be used to estimate semi-quantitatively how many slides

can be stained satisfactorily per given volume of hematoxylin or any other
stain for that matter.

• Since each of the three stains (HX, OG, EA) contains different concentrations
of dyes, you may find that more slides can be stained in one stain than
another, so that all three stains are not changed at the same time
 CYTOPREPARATION-STAINING

• Alcohol rinses: work best when clean, deep/a lot, and are used for sufficient
time to effect removal of excess dye from the Pap smears.

• Dirty rinses become more like a stain than a rinse and prevent effective
rinsing.
 CYTOPREPARATION
• Cross-Contamination Control
• Filtration: To prevent cross contamination all stains, fixatives, rinse
alcohols and xylene should be filtered to remove floaters using
recommended Millipore filter paper

• Cross-contamination prevention measures include

• (1) avoiding undue agitation (e.g., running water, rapid dipping),
• (2) frequent replacement of non-stain solutions,
• (3) staining GYN specimens before non-GYN preparations
 CYTOPREPARATION
• Cross-Contamination Control

• (4) Staining high-risk shedder specimens (e.g., body cavity fluids) last,

• (5) Maintaining a separate Pap stain set-up for non-gyn specimens,

• (6) Staining known shedders in separate miniature Pap stain set-ups—using

nearly exhausted solutions that can be discarded afterwards where
appropriate, and

• (7) Filtering xylene or xylene substitute baths through laboratory grade

filter paper to remove particulates.
FILTRATION
 COVERSLIPPING

• Use small amount of mountant and thin cover glasses

• Cover-glass-side-down can still be seen under 10×, and it is not until trying to
focus under 40× that one realizes the slide is upside-down.

• Freshly mounted preparation looks sharper under 10× than it does under 40×
and that older preparations look sharp under all magnifications.
• These examples demonstrate the fact that objectives lenses are sensitive to
deviations in mounting medium and cover glass thickness as a function of their
numerical objectives.
• Deviations will not be visible under 10× objectives, as their numerical aperture
of 0.25 make them insensitive to cover glass thickness.
 COVERSLIPPING

• Objectives with numerical apertures equal to or greater than 0.6 (i.e., 40×)
require a cover glass thickness of 0.180 mm (i.e., No. 1-1/2 thickness).

• Freshly mounted preparations rarely have the optimum thickness of

mounting medium and cover glass.

• The proactive tactic, therefore, is to use cover glasses, which are thinner,
and as little mounting medium as is consistent with a permanent mount.

• Some mounting media cause biological dyes to fade even when stored in
the dark, Test all new batches of mounting medium for fading
 MICROSCOPY
• Use clean microscopes illuminated according to the Principles Of Köhler

• 1. Focus sharply on the cells of interest throughout the steps that follow.

• 2. Close the field diaphragm until it is slightly smaller than the field-of-view.

• 3. Center the field diaphragm within the field-of-view so that the circumference
of the illuminated field is equidistant from the outermost circumference of the
field-of-view.
•
• 4. Close the field diaphragm to its smallest opening. (One could attempt to
center it at this step, but centration is difficult to judge.)
 MICROSCOPY
• 5. Close the aperture diaphragm to its smallest opening.

• 6. Raise or lower the substage condenser until the edges of the field
diaphragm are in sharp focus while the cells are also in focus.

• When in focus, the edges of the field diaphragm will be ringed by a magenta
halo and the top lens of the substage condenser will be 1 to 2 mm from the
underside of the slide, nearly touching it.
•
 MICROSCOPY
• When the substage condenser is raised too much, the halo will be orange;
when lowered too far, blue. the intensity of the colors will vary with the
opening of the aperture diaphragm. “Raised too much” and “lowered too
much” occur within a distance of one to two millimeters of the optimal
height.

• 7. Open the field diaphragm until the edges just disappear from view.

• 8. Open the aperture diaphragm until best contrast is obtained.