 Accurate cytologic Interpretation of cytological

specimen depends on
1.Method of specimen collection

2.Fixation & Fixatives

3.Presrvation of fluid specimen prior to

processing
4.Preparation of material for microscopic
examination.

5.Staining & mounting of the cell sample

 The Most commonly used procedures are
1.Direct or sediment smear on glass slide

2.Cytocentrifuge preparation

3.Preparation with membrane filter

4.Preparation of cell blocks

 Petri dishes& brown paper toweling
 Curets, applicator sticks, forceps,
 Mayer’s albumin (adhesive)
 Copper paper clips for keeping slides
separated in fixative
 Glass slide
 Bottles of 95% ethyl alcohol.
 Centrifuge tube
 Centrifuge
 Mix by stirring 1 volume of fresh egg white or
reconstituted dried egg albumin(1g of albumin per
20 ml DW)
 Add equal volume of glycerol to it.
 Filter this mixture using coarse filter paper in an
oven (550Cto580C)55
 Add few crystals thymol to prevent growth of
molds.
 Store this solution in a screw capped bottle at a
temperature of 20C.
 Arrange the slide on a clean tray.
 Using a dropper place a drop of Mayer’s albumin
on the slide.
 Spread the albumin thinly & evenly on the slide.
 Lay another slide on the top ,rub the two slides
together to obtain a uniformly coated slides.
1. Gelatin chrome alum
Gelatin-1gm
Chrome alum-0.1gm
Contents are dissolved in 100ml DW. To which
1ml of 10% thymol is added.
slides are dipped once, drained & their backs
wiped off & the allowed to dry.
2. Poly –L-Lysine
Used with scanning electron microscopy

3. 3 aminopropyl triethoxysilane
Used for DNA in situ hybridization

4. Elmer’s Glue
 Used for specimen consisting of small amount of
material.
 Eg: Cervical scrapes, brushing, needle aspirates.
 Symptomatic patients

 Techniques:
 –”pick and smear”
 – Saccomanno
 – Cytospin or monolayer
 Fresh or Prefixed in 50 % ALCOHOL
select proper particle for examination.
observe for bloody, discolored or solid particles.
 Place a small portion of particle on plain slide.
 With a clean glass slide crush the particle of
sputum with a rotary motion.
 With overlapping horizontal stroke spread the
material evenly on the slide.
 Place the smear immediately in 95% ethyl alcohol
fixative.
 In the absence of particles, sputum sample from at
least four different portion must be smeared.
Procedure:
1.Place the specimen received in Saccomanno’s
fixative ,in 50% ethanol & make up the volume
up to 50 to 100ml.

2.If specimen is received in 50% alcohol add

sufficient amount of saccomanno’s stock solution.
3.Pour the specimen into container and blend in a
blender at high speed up to 10 to 15 seconds
4.Pour the blended specimen in to a 50ml test
tube.
5.Centrifuge the specimen for 10 minutes.
6.Decant the supernatant, leaving few drops of
fluid to mix with sediment.
7.Resuspend the sediment by agitating the tube
on a vortex mixer.
8.Place few drops of the sediment in the center of
clean slide.
9.Place a second slide over the material and allow
it to spread evenly between the two slides.
10. Gently pull the slide a part with easy sliding
motion.
11.Allow the slide to air dry. Until ready for
staining
 As with coating fixative the slides should be
rinsed in 95% alcohol for at least 10minutes before
staining.(to remove carbowax)
 Voided or catheterized urine specimens can be processed for microscopy
by one of the following methods.
Centrifugation followed by cytocentrifugation:
Alcohol fixation is necessary before staining with the
Papanicolaou method. Cytocentrifugation should produce a sample
which is almost a monolayer of cells measuring about 6 mm in
diameter.

 Direct smears. They are easier to prepare but show usually scanty
cellularity.
 Membrane filter preparations. They show very good cellularity, but
are difficult to prepare. Preparations are fixed in alcohol and stained
using the Papanicolaou stain.
 Monolayer preparations such as ThinPrep (Cytyc Corporation,
Boxborough, MA, USA) are also used to prepare urine samples. The
preservation of the cells is usually very good and the procedure is
easier than the filter method. The cost, at the moment, is probably the
main limitation here.
 Fixation
1-12hr, no fixation needed
12-24hr, refrigeration
24hr or more, fixation with equal volume of 50-
70% alcohol or carbowax
Transport: 1:1 dilution with ethanol 50%
 Slide Preparation
Sedimentation and smearing
Membrane Filtration
Cytocentrifugation
Cell Blocks
Method Advantages Disadvantages

Cytocentrifugation Simple, large-sized clusters, Air-drying artefact; multiple slides

better-preserved need to be prepared due to cell
architecture loss; more unsatisfactory or
less than optimal specimens
Membrane Filter Good morphology Difficult to prepare, rapid drying makes storage
difficult, cells are distorted by pores, cells that are
placed in various planes of focus makes screening
tedious, background may not be clean, requires
fresh specimens as prefixation coagulates proteins
that clog the filter; longer screening time

Thin Prep (TP) Standardized and easy preparation,

Standardized and monolayer, Some alteration of key nuclear
increased cellularity, better morphologic features and
preservation, background elements; fragmentation
decrease in less-than-optimal
of cell clusters, cell shrinkage;
specimens,
more expensive than conventional preparations
uniform cell distribution, clean
background,
shorter and easy screening, multiple
slides can be prepared, additional cost
is offset by improved specimen
quality
SurePath (SP) Standardized preparation, excellent Cells that are in various planes of
cell yield, focus make screening and
preservation and morphology, focusing at high magnification
multiple slides can be prepared, slides tedious
are stained on the processor
 Accuracy
 Sensitivity, Moderate
 Positive rate 25%-72%
 Increases if “suspicious” included
 Multiple urine samples increases sensitivity
 Grade of urinary bladder tumour

 Specificity, High
 95%-100%
 False positive seen in bladder stones, polyoma virus, chemotherapy
 FRESH SPECIMEN
Pour specimen in to 50ml centrifuge tube&
centrifuge for 10 minutes.
Pour off the supernatant
Transfer the sediment to a clean glass slide.
 Three methods are mainly used for preparation of the
slide.
A. Place two drops of sediment on a slide using glass
pipette.

Place a clean second slide over the sediment &allow

to spread evenly between the two slides.

Gently pull the slide apart wit a sliding motion.

Fix immediately in 95% ethyl alcohol
B.
 Dip a thin tightly wound cotton swab
premoistened with supernatant in the sediment
 Gently roll the swab in one direction over the
surface of the slide & immediately fix it
C.
 Touch the bacteriology wire loop to the sediment
 Move the loop quickly in a longitudinal, then
horizontal direction over the surface of the slide.
 Immediately fix it
 Cell block technique or paraffin embedding of
sediments of fluids is among the oldest method of
preparing material for microscopic examination.
Advantage
 Multiple sections of the same material may be
processed for routine stain such as haematoxylin
&Eosin &special stain such as stain for mucin, melanin
, Bacteria & fungi
 Recognition of histologic pattern of disease that some
time cannot be readily identified in smears of filter
preparation
 Sputum,effusions, urine sediment &material from GIT
are suitable for cell block preparation
 Mix the sediment or tissue fragment in fixative
recommended for cell blocks
 If the sediment is bloody ,the blood may be
haemolysed prior to the addition of fixative
 Centrifuge this mixture for 10 minutes& let stand for
overnight without disturbing the sediment
 Pour off the supernatant & drain the tube well by
inverting it on a paper towel
 Carefully remove the packed sediment or fibrin clot
from test tube using spatula and wrap in lens paper
 Place wrapped sediment carefully in labeled tissue
cassette.
 Put the tissue cassette in to jar containg fixative &
process as tissue
 Mix the sediment or tissue fragment in fixative
recommended for cell blocks
 If the sediment is bloody ,the blood may be
haemolysed prior to the addition of fixative
 Centrifuge this mixture for 10 minutes& let stand for
overnight without disturbing the sediment
 If the sediment becomes hard & packs well, gently
remove it from the test tube with spatula& place it
conical side up & slice the sediment using scalpel
 Place the cut side of sediment in a melted agar that has
been spread on petridish & allowed to harden
 Trim excess agar from sediment & place button in
tissue casette.
 Put it in jar containing fixative& process as tissue
 Thoroughly mix a few drops of outdated blood
plasma with the fresh unfixed sediment
 IF it is fixed priorly it should be washed with
balanced salt solution for several time.(alcohol
inhibit clotting action of plasma & thrombin)
 Add equal volume sediment containg plasma &
thrombin ,mix well. This will form a clot.
 Place the clot in a cassette that has been lined with
lens paper to prevent the clot from oozing through
the hole.
 Put the tissue cassette into jar containing fixative&
processed as tissue
 Introduced by Korgerus & Anderson-1988
 Ensure minimal cell loss.

Procedure
 In a 50mL plastic conical centrifuge tube fix cell sample
with 50% alcohol for 1hour
 Spin sample at 300g for 7 minutes & pour off supernatant.
 Resuspend the cell pellet in 3ml acetone for 10 minutes
 Spin sample for 3 minutes. Pour off acetone.
 Place tube in a warm plate (not more than 60oC)
 Add melted paraffin to the dry warm pellet
 After solidification of paraffin tap the bottom of tube to
remove block
 Cut & process the conical end of paraffin block like any
tissue section
 Applied for slide poorly stained,slide that faded
because of age=or when special stains are desirable.
 To remove cover slip
 Soak in xylene until cover falls off or heat on a
warming plate at 60oCfor 3to 4hrs
 Place the slide in freezer with cover slip down for a fw
minute to half an hour.
 The cover slip should separate from the slide
 Soak the slide in xylene to remove old mounting media
 Rinse slide well in 95% ethanol& water for two or
three times this will remove all counter stain
 Place the slide in aqueous 0.2% to 5% solution of
HCl or 1% HCL in 70% alcohol for 5 minutes to 1
hour to remove haematoxylin.
 Remove acid by rinsing in running tap water for
10 to 15 minutes , to ensure complete removal
slide should be place in Scott’s tap water & rinsed
again in tap water.
 Slides are now ready for restraining
 Mounting medium creates permanent bond
between the slide & cover slip.
 The permanent bond protects the cell from
mechanical damage, air drying effect& stain
fading.
 Slide can be stored permanently
 Regardless of the mounting medium used it is
important to maintain its pH close to neutral as
possible to prevent the fading of the stain.
 1gm of 2,6-di-tert-butyl-p-cresol can be added to
100ml of mounting medium to inhibit fading of
the stain
 Coverslip
 No.1 cover slip in size 24x 50mm are used.
 Cover slippig the cell sample
 Well mounted slide should be free of air bubbles
&artifacts.
 A minimum of mounting medium should be used
 Too much mounting medium interfere with
microscopic details, Making the film hazy or
milky.
 Remove slide from xylene
 Place two drops of mounting medium on glass
slide or coverslip.
 Lower the coverslip over the slide to which
mounting medium has been applied,
 Bubbles should be avoided
 Turn the slide right side up
 Gently tease bubble from under the cover slip with
an applicator stick & wipe excess xylene &
mounting medium from the slide