CYTOLOGY PROTOCOLS

CYTOLOGY PROTOCOLS
Table of Contents
SPUTUM PREPARATION
SPUTUM SMEARS
SPUUTUM (Blender method)
BRONCHIAL/GI BRUSHINGS
BRONCHIAL/GI WASHINGS

SPUTUM PREPARATION

A sputum series is the least invasive method for detecting pulmonary cancer. Patients
with a history of smoking, chronic obstructive
pulmonary disease or airflow obstruction,
long term exposure to uranium or asbestos, or a history of metastatic carcinoma are at a
higher risk for the development of tumor. A sputum series consists of one fresh specimen
collected the FIRST THING IN THE
MORNING for three consecutive mornings. The rate
of detection for pulmonary carcinoma improves when more specimens are
submitted. One
specimen may be a post bronchoscopy sputum specimen which is produced immediately after
the bronchoscopy
Procedure. This sputum should never be discarded as it may contain
valuable diagnostic material treed during the procedure. The
minimum volume of specimen
accepted is 1.0 ml, however the quality of sputum is Judged by its contents rather than
quantity. If a
series of consecutive early morning sputum specimens is collected and
submitted to the laboratory together as a complete group, they
may be pooled during
processing resulting in one final cytologic report.

SUPPLIES NEEDED

COPLIN JAR
WOODEN STICKS OR MICROSCOPE SLIDES
95% ETHANOL
TONGUE DEPRESSOR
BIOHAZARD HOOD

METHOD

1. Verify the patient name on the requisition and specimen container.

2. Complete a NONGYN form describing the specimen appearance in detail, number of slides
prepared, and stains performed.
3. Label 3 slides with the pathology accession number and last name of patient using lead
pencil.
4. Using wooden sticks or tongue depressor, select areas that appear opaque or bloody and
spread the material thinly and evenly
over / of the slide surface.
5. Immediately immerse 2 of the slides in coplin jar containing 95% alcohol and set aside
until ready for staining process (see
Staining Schedules).
6. Use a color coded slide and make a third smear, allow it to dry, then stain with DIFF
QUIT Give to cytotechnologist for
preliminary check. (See Staining Schedules>).
7. Record the date of preparation on the specimen container and refrigerate remaining
specimen. Specimen is stored for one week
before disposal.

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PROCEDURE: CYTOPREPARATION METHODS

SUPPLIES NEEDED FOR BLENDER METHOD

COPLIN JAR
MICROSCOPE SLIDES
WARING BLENDER
SACCOMANNO FIXATIVE
BIOHAZARD HOOD
BLENDER CUPS
50 ML CENTRIFUGE TUBES

BLENDER METHOD

1. Verify the patient name on the requisition and specimen container.

2. Complete a NONGYN form describing the specimen appearance in detail, number of slides
prepared, and stains performed.
3. Label 2 slides with the pathology accession number and last name of patient using lead
pencil.
4. Add 50 ml of Saccomanno fixative to specimen if none was added previously.
5. Shake thoroughly to mix.
6. Pour into blender cup and homogenize for 20 to 30 seconds.
7. Pour homogenate into prelabelled 50 ml centrifuge tube.
8. Centrifuge for 5 minutes at 1500 RPMs.
9. Decant all supernatant and mix on vortex for 15 seconds at High setting.
10. Place 2 or 3 drops of mixed concentrate on one slide.
11. Make smears by laying second slide over first allowing material to flow evenly until
of the slide surface is covered. Gently
slide apart and lay on a flat surface to dry.
12. When completely dried, immerse in 95% ethanol for 15 minutes before staining. (See
Staining Schedules)
13. Record the date of preparation on the specimen container and refrigerate remaining
specimen. Specimen is stored for one week
before disposal.

PROCEDURE NOTES

Ancillary Studies: If specimens are collected for culture and cytology, the sample
must go to Microbiology first. Detection of
Pneumocystis carini may be done on sputum;
however, bronchoscopic lavage is more successful in detecting the organism. If
Pneumocystis is requested, make an additional smear and submit to Histology for Grocott's
Methenamine Silver staining (GMS).

Sputum from the same patient which is collected on consecutive days may be poured
together or pooled.

If specimen is received in alcohol or Saccomanno fixative, make crush smears or use

blender method. Place a small quantity of
hardened material between two slides and crush
the material until it begins to coat the slide uniformly. Immediately immerse the
slides
in 95% alcohol.

If the specimen is watery, it probably represents saliva. Make direct smears if a cell
button is obtained after centrifugation. If not, use

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cytocentrifugation to prepare the

smears. Follow procedure for preparing brushing specimen. URINARY TRACT SPECIMENS:
(Voided
Urine, Catheterized Urine, Ureteral, Renal Pelvis, Bladder Washing, Urethral Specimens)

PROCEDURE: CYTOPREPARATION METHODS

BRUSHING SPECIMENS (Bronchoscopy and Gl tract)

Brushing specimens may be submitted from suspicious areas seen during bronchoscopy or
endoscopy.

SUPPLIES NEEDED

COPLIN JAR
SLIDES as needed
95% ETHANOL
3 SAMPLE CHAMBER ASSEMBLIES
PIPETS CYTOCENTRIFUGE
VORTEX MIXER
MICROSCOPE
BRUSH WASH VIALS
BIOHAZARD HOOD
SACCOMANNO FIXATIVE
BALANCED SALT SOLUTION

METHOD

1. Verify the patient name on the requisition and specimen container.

2. Complete a NONGYN form describing the specimen appearance in detail, number of slides
prepared, and stains performed.
3. Label 3 slides with the pathology accession number and last name of patient using hard
lead pencil.
4. Load 3 slides and sample chamber assemblies into the cytocentrifuge.
5. Brushings are usually collected in a Saccomanno Brush Wash vial that contains Saccomanno
fixative. Because the quantity is
small, precentrifugation is usually not needed. If the
brush is submitted, tease any remaining material from the brush by
pushing it back and
forth through special insert in the Brush Wash vial. If the brush is separate, add a few
milliliters of
Saccomanno fixative to the container and vortex it on high speed for 15
seconds.
6. Saccomanno fixative will change the structure of cells, so one must estimate the
cellularity visually. The solution should appear
slightly hazy. For specimens appearing
clear, the sample may be equally divided among three chambers. Saccomanno fixative
may
also be added to dilute specimens appearing cellular.
7. Load three sample chamber assemblies and spin for 5 minutes at 1000 RPMs.
8. Carefully remove sample chambers and clips as described earlier. Drying is permissible
due to the addition of Saccomanno
fixative and should occur in a few seconds. Immerse
slides in coplin jar containing 95% alcohol and set aside until ready for
staining
process. One slide may be stained with DlFF-QUIK for preliminary check. (See Staining
Schedules).

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9. Record the date of preparation on the specimen container and refrigerate any remaining
specimen. Specimen is stored for one
week before disposal.

PROCEDURE NOTES

Ancillary Studies: Brush specimens may be submitted in saline, although saline is a

poor

transport medium for maintaining cell morphology. Add an equal volume of Saccomanno
fixative

upon receipt in the Lab. This specimen must be centrifuged by pouring into a clean
prelabeled

centrifuge tube and spinning for 5 minutes at 1500 RPMs. Decant supernatant down to the
conical

portion of the centrifuge tube and follow instructions for visual estimation of
cellularity.

PROCEDURE: CYTOPREPARATION METHODS

If Microbiology studies are requested on the same specimen, it must go to Microbiology

first. Requests may be for fungal
identification or ova and parasites. Ask Microbiology to
return remaining specimen to cytology for processing or indicate this
information directly
on the requisition.

If Brush Wash vial is submitted with only a drop or two of fluid, use additional
Saccomanno fixative to thoroughly rinse the container
and brush. Use vortex mixer to
remove as much material as possible.

PROCEDURE: CYTOPREPARATION METHODS

WASHING SPECIMENS (Bronchoscopy and Gl tract)

Cytology specimens may be collected during bronchoscopy or endoscopy to augment the

biopsy sample. The surface of suspicious
areas should be aspirated, brushed and then
biopsied. If insufficient secretions are present, or if a lesion is not visualized, the
area in
question should be ravaged. Sometimes, rinsing the biopsy forceps in the saline
used for the washing will provide additional material
for examination. For full diagnostic
value, all material should be identified as to site of origin. The minimum acceptable
volume for
washing specimens is 1 ml.

SUPPLIES NEEDED

COPLIN JAR
SLIDES as needed
95% ETHANOL

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LABELED TISSUE CASSETTE

PIPETS CONTAINER with FORMALIN
VORTEX MIXER 3 SAMPLE CHAMBER ASSEMBLIES
50 ML CENTRIFUGE TUBE
CYTOCENTRIFUGE
BALANCED SALT SOLUTION
MICROSCOPE
SACCOMANNO FIXATIVE
BIOHAZARD HOOD

MIXING VIAL

M ETHOD

1. Verify the patient name on the requisition and specimen container.

2. Complete a NONGYN form describing the specimen appearance in detail, number of slides
prepared, and stains performed.
3. Label 3 slides with the pathology accession number and last name of patient using hard
lead pencil.
4. Pour specimen into 50 ml centrifuge and spin for 5 minutes at 1500 RPMs.
5. If specimen quantity is sufficient, make a cell block. Carefully remove the cell
button without breaking it apart, wrap it in
tissue, and enclose it in a tissue cassette.
Place the cassette in formalin. Send cassette to grossing room for processing and
paraffin
embedding.
6. If material Is insufficient for cell block make 2 smears by placing 1 or 2 drops of
specimen on a slide. Lay the second slide on
top, allow the material to spread uniformly
until it covers of the slide surfaces, and gently slide them apart. Immediately
immerse
both slides in 95% alcohol.
7. Use vortex mixer or a pipes to thoroughly mix remaining sample concentrate.
8. If sample concentrate appears thick, dilute with several drops of balanced salt solution
or reserved supernatant until the
sample concentrate appears hazy.
9. Prepare 3 sample chamber assemblies and load in Cytospin. Use a color coded slide for
DIFF-QUIK staining. Add 6 drops of
concentrate to each sample chamber. For slides that
will be stained with Papanicolaou and Hematoxylin/Eosin, add 4 to 5
drops of Saccomanno
fixative.
10. Spin for 5 minutes at 1000 RPMS.
11. Remove the alcohol fixed slides first. Remove the sample chamber assemblies from
cytocentrifuge and carefully unlock slide
clip. Peel off blotter being careful not to
disturb cell button.
12. For Papanicolaou and H&E staining immediately immerse the slides in a coplin jar
containing
13. 95% ethanol and set aside until ready for the staining process (See Staining Schedules).
Remove the color coded slide last.
When dry, stain with DIFF-QUIK (See DIFF-QUIK Staining
Schedule)
14. Give to cytotechnologist for preliminary exam/ check for cross contamination potential.
If the cytotechnologist makes any
recommendations, adjust the number of drops as indicated
and make new cytospins. Discard the first set of slides.
15. Record the date of preparation on the specimen container and refrigerate remaining
specimen. Store specimen for one week
before disposal.

PROCEDURE NOTES

Ancillary Studies: If specimens are collected for culture and

cytology, the
sample must go to Microbiology first.

Detection of Pneumocystis carinii is more successful on bronchoscopic ravage. If

requested, make additional smear for Grocott's
Methenamine Silver (GMS) staining as
described for alcohol-fixed slides. Or if a cell block is obtained, cut an additional
section for

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GMS staining. GMS staining is performed in Histology.

Specimens that are received in alcohol cannot be used for Microbiology.

Specimens received in formalin will be prepared for cell block only. No additional
cytologic smears will be made.

If specimen is received in alcohol or Saccomanno fixative, make crush smears. Place a

small quantity of hardened material between
two slides and crush the material until it
begins to coat the slide uniformly. Immediately immerse the slides in 95% alcohol.
Alternatively, use blender method for 15 seconds to homogenize specimen. (See Sputum).

To estimate cellularity of fresh unfixed specimens, place 1 drop of specimen on a slide

and coverslip. Rack the condenser knob down
and examine the slide. Select a representative
area of cells and switch to the high power objective. To achieve the optimal dilution,
there should be 10 cells per high power field. If there are too many cells, add more
balanced salt solution or reserved supematant. If
there are too few, recentrifuge the
specimen and remove more of the supernatnant.

Specimens containing mostly red blood cells may be diluted to 20 cells per high power
field.

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