Collection of Non-Gynecologic Specimens for Cytopathology Testing
• Specimen containers may be picked up in room A2-412, Cytology Lab. Fluid
should be poured immediately into the container.
Write patient's complete name, date of birth and/or medical record number on the
container.
• All specimens must be treated as potential infectious material. Therefore use
specimen transport bag. Place the completed cytology requisition in the outer
pouch. Place the specimen inside the bag. Refrigerate specimen if delivery is
delayed.
Collection of Sputum, Bronchial, Esophageal & Gastric Specimens
Sputum
• A deep cough specimen should expectorate directly into a jar containing CytoLyt
Fixative. Three specimens over 3 days are recommended (can be pooled). It is
crucially important to get a deep cough specimen, explain to the patient the
difference between sputum and saliva. Aerosol inhalants can be used to stimulate
sputum production. Obtain samples before meals to avoid food contamination.
Patient should thoroughly rinse their mouth before expectorating.
• Do not use 95% ethanol. Higher concentration of ethanol binds with
proteinaceous material to form a hard lump, which makes it difficult to mash up
and make thin-layered slides.
Bronchial Aspirates and Washings including Bronchoalveolar Lavage
(BAL)
• Bronchial secretion aspirate must be fixed immediately with an equal part of
CytoLyt Fixative. Rinse aspiration tube 2 or 3 times with a small amount of
CytoLyt and add these washings to the specimen container. Bronchial irrigation
with saline may be used. Fix as above. Specimens must be labeled whether
bronchial secretion or bronchial washing and from which side obtained.
Bronchial Brushings
• Brush should be appropriately handled to obtain adequate sampling and removed
from the bronchus with the entire bronchoscope so material is not lost from
surface of the brush. For each sample the entire scope and brush should be
reinserted. The entire sample and disposable brushes must be submerged in a
centrifuge tube containing CytoLyt fixative and delivered to the Cytopathology
lab. (A2-412). When cultures are indicated, send separate specimens to
Microbiology in the correct media.
Note: If there is a suspicion that the patient may have lipoid pneumonia, the sputum or
BAL should be collected fresh, unfixed for Oil Red O Fat Stain.
Esophageal and Gastric Washings
• Esophageal and gastric washings should be mixed with an equal part of CytoLyt
Fixative. Rinse aspirator with a small amount of fixative and add to the specimen
container.
Esophageal and Gastric Brushings
Esophageal and Gastric brushing are similar to bronchial brushings, above.
• The entire sample must be deposited into a centrifuge tube containing Cytolyt
fixative and delivered to Cytopathology Lab (A2-412). In certain clinical
circumstances, it may be appropriate to air dry slides. This option must be
discussed prior to specimen collection, with the Cytology Supervisor or Attending
Cytopathologist by calling the Cytology office at x 1666.
Exudates (Pleural, Pericardial and Peritoneal Fluids)
Pleural, Peritoneal, Pericardial and other Fluids
• Liquid body cavity fluids may be sent fresh and unfixed provided they are
promptly (1–2 hours after collection) delivered to the laboratory. All liquid body
cavity fluids should be kept refrigerated at all times, unless submitting
immediately to the Cytology laboratory for processing. DO NOT Freeze. If the
specimens are refrigerated immediately they can last over a 4-day-long weekend.
• For the best cytological morphology, NEVER add any kind of fixative or
anticoagulant. If a delay in processing is anticipated or refrigeration is not
possible, mix with equal volume of Cytolyt fixative and send to the laboratory.
• Do Not use high concentrations of ethanol as a fixative. 70% and 95% ethanol
will combine with protein in fluid to form heavy precipitation. The heavier the
precipitation, the more protein and fewer cells will be on the smears.
Spinal Fluid
• It is best to process CSF in the fresh state. During normal operating hours, fresh
CSF, ideally 5-10 ml can be brought directly to the laboratory for immediate
processing. During nights and weekends (or if specimen is not delivered
immediately to the laboratory) it should be fixed in equal volume of CytoLyt
Fixative and refrigerated.
• Volume: at least 1 to 2 ml if possible. Absolute minimum: 0.5 ml.
• Due to small volume, spinal fluids are stored in small test tubes.
• CSF Delivery - Please deliver CSF specimen to the lab as soon as possible. Cells
in CSF are very delicate and may degenerate if not processed immediately.
Fine Needle Aspirate (FNA) Specimens
• In general, for FNAs performed by clinicians, the preferred collection technique
regardless of the site of FNA is to deposit and rinse the entire sample into a
centrifuge tube containing Cytolyt fixative and deliver to the Cytopathology Lab
(A2-412) for processing. Direct smears made by clinicians (or with the assistance
of cytotechnologists or pathologists) may be applicable to certain specimens. This
option must be discussed, prior to sampling / specimen collection, with the
Attending Cytopathologist by calling the Cytology office at x 1666.
Urine and Upper Tract Brushings and Washings
Voided Urine
• Voided urine should be obtained 3-4 hours after the patient has last urinated.
• Recommended Volume: 25-100ml.
• Collect urine in a clean container, mix well and put lid on tightly. Deliver to the
doctor’s office, clinic or Cytology Lab promptly.
• If a delay in transport to the lab is anticipated, refrigeration is recommended.
• If a delay of more than 24 hours is anticipated, fixation with approximately an
equal volume of Cytolyt fixative is recommended to avoid degeneration.
Note: DO NOT drink water to induce urination within an hour or so. Urine collected by
this method produces poor cellular content.
Catheterized Urine
• This is the method of choice for female patients. If malignancy is suspected,
voided urine from a female patient has no value in establishing tumor
involvement of the bladder. Malignant cells from the vagina cannot be ruled out.
• Midstream urine is an alternative method of collecting urine if catheterization
technique is not available.
• To obtain midstream urine: Wash vaginal area well. Urinate approx half of the
urine from the bladder. Collect the second half of the voided urine in a clean
container. Mix well.
• Catheterized urine collection is also used by the Urologist during the Cystoscopic
procedures (i.e. bladder washings). Volumes may vary, in part depending how
much urine is in the bladder at the time of Cystoscopy.
• Recommendations for refrigeration and fixation are as given above for voided
urine specimens.
Ureteral / Pelvic Brushings and Washings
• The entire sample from Ureteral and Pelvic brushings should be submerged in a
centrifuge tube containing CytoLyt fixative. For washings, ratio of specimen to
CytoLyt fixative should be close to 50/50.
• Recommendations for refrigeration and fixation are as given above for voided
urine specimens.
Collection of Gynecologic Specimens for ThinPrep Pap test
Purpose
The detection of cervical cancer and its precursors as well as other Gynecologic
abnormalities is the primary purpose of obtaining a cervical cell sample. It is important to
obtain a specimen that is not obscured by blood, mucus, inflammatory exudates or
lubricant.
The ThinPrep 2000 System is used in the processing of liquid-based Gynecologic
specimens - ThinPrep Pap Test. In conjunction with the Molecular Pathology Laboratory
at DMC, HPV testing is also performed on patients age 30 and older, and, reflexively, on
younger patients with PAP results of ASCUS. The HPV testing results are posted
separately in the Cerner System at DMC.
The Procedures
Preparation of Patient
• The patient should be tested 2 weeks after the first day of her last menstrual
period and definitely not when she is menstruating. Even though ThinPrep
reduces obscuring blood, clinical studies have demonstrated that excessive
amounts of blood may still compromise the test and possibly lead to an
unsatisfactory result.
• The patient should not use vaginal medication, vaginal contraceptives, or douches
during the 48 hours before the exam.
Specimen Collection Preparation
• Lubricant jellies should not be used to lubricate the speculum.
• Remove excess mucus or other discharge present before taking the sample. This
should be gently removed with ring forceps holding a folded gauze pad. The
excess cervical mucus is essentially devoid of meaningful cellular material and
when present in the sample vial may yield a slide with little or no diagnostic
material present.
• Remove inflammatory exudates from the cervical canal before taking the sample
by placing a dry 2x2 inch piece of gauze over the cervix and peeling it away after
it absorbs the exudates or by using a dry proctoswab or scopette. The excess
inflammatory exudates is essentially devoid of diagnostic cellular material and
when present in the sample vial may yield a slide with little or no diagnostic
material present.
• The cervix should not be cleaned by washing with saline or it may result in a
relatively acellular specimen.
• The sample should be obtained before the application of acetic acid.
Collect Gynecologic Sample Using the Broom–Like Device
• Insert the central bristles of the broom into the endocervical canal deep enough to
allow the shorter bristles to fully contact the ectocervix. Push gently, and rotate
the broom in a clockwise direction five times.
• Rinse the broom as quickly as possible into the PreservCyt Solution vial by
pushing the broom into the bottom of the vial 10 times forcing the bristles apart.
As a final step, swirl the broom vigorously to further release material. Discard the
collection device.
• Tighten the cap so that the torque line on the cap passes the torque line on the
vial.
• Record the patient’s name, date of birth and/or medical record number on the vial.
• Record the patient information and medical history on the cytology request form.
• Place the vial and requisition in a specimen bag for transport to the laboratory.
Collect Gynecologic Sample, Using the Endocervical Brush/Spatula Device
• Obtain an adequate sampling from the ectocervix using a plastic spatula with 360
degree rotation while maintaining tight contact. Rinse the spatula as quickly as
possible into the PreservCyt solution vial by swirling the spatula vigorously in the
vial 10 times. Discard the spatula.
• Obtain an adequate sampling from the endocervix using an endocervical brush
device. Insert brush into cervix until only the bottom-most fibers are exposed.
Slowly rotate ¼ or ½ turn in one direction. Do not over rotate.
• Rinse the brush as quickly as possible in the PreservCyt Solution by rotating the
device in the solution 10 times while pushing against the PreservCyt vial wall.
Swirl vigorously to further release material. Discard the brush.
• Tighten the cap so that the torque line on the cap passes the torque line on the
vial.
• Record the patient’s name, date of birth and/or medical record number on the vial.
• Record the patient information and medical history on the cytology requisition
form.
• Place the vial and requisition in a specimen bag for transport to the Laboratory.
Collection of Conventional PAP Smears
The Procedures
• Preparation of patient – the same procedures as in 01.8.1
• Print patient's name at the frosted end of the slide before collecting specimen.
• The cervical OS, transformation zone and any lesion must be sampled. Cytobrush
is preferred device for sampling. After the smear is made, immediately spray the
slide with spraycyte. Follow instructions on the spray container. Spray
thoroughly. Smear should still be wet when spray is applied to prevent cellular
deterioration.
• If spraycyte is not available, put smear in container containing 95% ethanol
fixative immediately.
• Put sprayed slides in slide cardboard holders. Allow smears to dry before putting
smears in slide folders to prevent cells sticking to inside cover.
• All specimens are classified as potential infectious material. Therefore use
specimen transport bag.
• Record the patient information and clinical history on the Cytology requisition
form. Put the cytology form in the outer pouch. Place the specimen in the inside
pouch. Deliver specimen to the Lab for processing.
Specimen Delivery
• Cytopathology Lab specimen processing room A2-412, 9 am to 5 pm, Monday
through Friday.
• Central Accessioning room A2-428 for Evenings, weekends, and holidays.
Specimen Container Pick-Up
• Cytopathology Lab Processing Room A2-412, 9 am to 5 pm Monday through
Friday.
• The refrigerator in the vicinity of Central Accessioning room A2-428 - Cytolyt
fixative available for Non-Gynecologic samples and FNA specimens. (if needed
for evenings, weekends, and holidays)
References
• Koss, LG, "Diagnostic Cytology and its Histopathologic Bases" 5th edition. 2006
• "Papanicolaou Technique; Approved Guideline” NCCLS Document GP15-A Vol.
14 No 8.
• ThinPrep 2000 System Operator’s Manual: Chapter Four (4.1-4.6).
• Cibas, ES. “Cytology Diagnostic Principles and Clinical Correlates” 2nd edition.
2003