Cytology

Introduction

The purpose of a cytological examination is generally for the cytological detection of malignancy. The Cytology Laboratory offers screening of cervical-vaginal samples and the interpretation of non-gynecological and fine needle aspiration (FNA) specimens with the ultimate goal of attaining the most accurate and complete diagnosis possible.

The goals of the service include:

• To maintain excellence in specimen preparation and diagnostic accuracy
• To provide the highest quality consultative services to our clinical colleagues, both within our institution and elsewhere
• To train residents, fellows, medical students and cytotechnologists in diagnostic cytopathology
• To support and incorporate advanced diagnostic techniques to improve diagnostic accuracy

Range of Laboratory Services

Services provided by the laboratory include:

• Gynaecological Cytology (Conventional Cervical Pap Smear and Liquid-based Gynaecological Cytology)
• Non-Gynaecological Cytology (e.g., sputum, urines, peritoneal/ pleural fluid, lavages)
• Fine Needle Aspiration Cytology
• Identification of infections and associated organisms including bacteria, fungi, protozoa and viruses. Special stains may be used to aid in the detection of these organisms.
• Evaluation of joint aspirates for crystals.

Other services available :

• One-Stop Thyroid FNA Cytology Clinic 
  Available every Tuesday (morning only) and Wednesday within Outram Campus. Cases will be reported within a few hours of receipt. 
  Please contact the laboratory for details.
• One-Stop Breast FNA Cytology Clinic
  Available every Thursday within Outram Campus. Cases will be reported within a few hours of receipt. 
  Please contact the laboratory for details.
• FNA Procedure Performed by a Pathologist (referrals accepted from GS & ENT currently)
  Available Monday to Friday (except PH); 10am to 4pm, SOC K20. 
  Booking is required. Please call 8125 3479 to schedule an FNA appointment.
  Otherwise, please call the cytology lab @ 6321 4954 for any enquiries.
• EUS/ EBUS FNA - Cytotechnical Assistance
  Available every Tuesday, Thursday at Endoscopy Centre, Block 6, level 2.
• Radiologically Guided Biopsy/Aspiration- Cytotechnical Assistance 
  Available every Tuesday and Thursday afternoon at Diagnostic Radiology Department

Special Instructions on Specimen Collection and Handling

Some cytological specimens require immediate fixation while others need to be fresh/unfixed specimens. All fresh/unfixed specimens should be sent to the laboratory immediately, if possible.

CSF will degenerate within one hour, even with refrigeration. It should be sent by hand to the Cytology Lab as soon as possible.

All samples must be clearly labelled with the patient’s name and unique identification number, e.g. NRIC number. In the case of prepared smears, all glass slides must be clearly labelled with the patient’s NRIC number.

All tests requested e.g. detection of TB, fungi, crystals, immunohistochemistry, cell block preparation etc, must be clearly indicated on the form.

Please contact the Cytology Lab at 6321 4954 for urgent specimens that require same day reporting. Urgent cytology specimens should be indicated on the request form and delivered immediately by hand to the Cytology Lab.

Please refer to following sections for site specific collection instructions:

• Cervical (Pap) Smear
• Non-Gynaecological Smear, Fluid and Fine Needle Aspiration (FNA)

Gynaecological Cytology - Cervical (PAP) Smear

The cervical (PAP) smear is a screening technique to aid in the detection of cancer and cancer precursors of the uterine cervix. It is not a diagnostic procedure. Both false-positive and false-negative results have been experienced with PAP smears. Accordingly, any lesion detected on screening should be biopsied. The PAP smear should not be used as the sole means to diagnose or exclude malignant and premalignant lesions.

1. Test Request Form

The request form must include:

• Patient’s name, age and identification number
• Submitting physician's name and contact number
• Date of specimen collection
• Source of material submitted (cervical, endocervical, vaginal, other body site)
• Last menstrual period (LMP)
• Hormonal status (e.g. post-menopausal, gravid)
• Exogenous hormone therapy (including birth control pills, treatment for endocrine-responsive malignancy, estrogen creams)
• Use of intrauterine device (IUD)
• DES exposure
• History of abnormal cytology and gynaecological disorders
• History of systemic chemotherapy, pelvic radiotherapy, gynaecologic surgery, cryosurgery, electrocautery, or laser surgery
• Any current abnormal clinical findings or patient symptoms; and
• Risk factors for cervical cancer (e.g., multiple sexual partners, sexually transmitted diseases including human papillomavirus [HPV], sexual activity at an early age, and smoking) if obtainable.
• Date of last gynaecological smear, if any

It is imperative that these instructions be strictly adhered to, omission of which may result in delay of reporting.

2. Patient Preparation

• Ideal sampling date is two weeks after the first day of the LMP
• Discourage sampling during normal menses
• Avoid use of vaginal medication, vaginal contraceptives, or douches for 48 hours prior to examination.

3. Collection of Specimen: General Consideration

Successful methods of specimen collection include the following:

• Before sample collection, label the frosted end of the glass slide with the patient's initial and NRIC number. The identifiers should be legibly printed on the frosted end of the slide using a hard lead pencil (e.g., 2B). If the specimen consists of more than one site, it is mandatory that the source of the specimen be indicated.
• It is important to obtain a smear that is not obscured by blood, mucus, or inflammatory exudate.
• During smear taking, clinician should wipe away excess mucus plug at the cervical os with ring forceps holding a folded gauze pad.
• Inflammatory exudate may be removed by placing a piece of gauze over the cervix and peeling it away after it absorbs the exudate.
• Visually inspect the cervix for abnormalities. Identify the transformation zone, if visible, and direct sampling efforts to encompass this area.
• If there is overlying mucus and exudate or necrotic material, these should be removed prior to sampling underlying lesion.

4. Collection Procedure

(A) Liquid-Based (ThinPrep) Gynaecological Cytology
(a) Broom-Like Device Protocol - extracted from Hologic Publication

1. Obtain an adequate sampling from the cervix using a 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.

2. Rinse the broom 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.

3. Tighten the cap so that the torque line on the cap passes the torque line on the vial.

4. Record the patient’s name and ID number on the vial and 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.

Note: Under no circumstances should the speculum be lubricated with medical jelly as it can make the Pap unsatisfactory.

(b) Endocervical Brush/ Spatula Protocol - extracted from Hologic Publication

1. Obtain an adequate sampling from the ectocervix using a plastic spatula.

2. Rinse the spatula into the PreservCyt Solution vial by swirling the spatula vigorously in the vial 10 times. Discard the spatula.

3. Obtain an adequate sampling from the endocervix using an endocervical brush device. Insert the brush into the cervix until only the bottommost fibers are exposed. Slowly rotate ¼ or ½ turn in one direction. DO NOT OVER-ROTATE

4. Rinse the brush in the same vial of PreservCyt Solution by rotating the device in the solution 10 times while pushing against the PreservCyt vial wall. Swirl the brush vigorously to further release material. Discard the brush.

5. Tighten the cap so that the torque line on the cap passes the torque line on the vial.

6. Record the patient’s name and ID number on the vial and 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.

Note: Under no circumstances should the speculum be lubricated with medical jelly as it can make the Pap unsatisfactory

(B) Conventional Cervical Pap Smears
(a) Spatula Protocol

1. Choose the contoured end of the spatula that best conforms to the anatomy of the cervix and the location of the transformation zone. A plastic spatula is recommended.
2. Rotate the spatula at least 360° about the circumference of the cervical os and ectocervix, while maintaining firm contact with the epithelial surface. (see Figure 1) (A clockwise rotation beginning and ending at 9 o’clock (or counter-clockwise rotation from 3 o’clock to 3 o’clock) will position the spatula so that the collected material is retained on the upper horizontal surface as the instrument is removed.)

   Figure 1. Sampling of the Cervix with Three Different Instruments: Spatula, Brush, and “Broom.”

3. Immediately spread the material collected evenly over the glass slide with a single, smooth stroking motion along the entire length of the slide within a few seconds (see figure 2 -4).
4. Apply spray fixative immediately (see figure 6). Allow fixative to dry before closing the slide container. Alternatively, specimen may be fixed in 95% alcohol.

   Figure 2.To transfer material from the spatula, smear the sample with a single stroking motion using moderate pressure to thin out clumps of cellular and mucus material. Avoid excessive force or manipulation, which will damage cells.

   Figure 3. To transfer material from the brush, roll the bristles across the slide by rotating the brush handle.

   Figure 4. To transfer material from the broom, smear the sample with a painting action, using both sides of the broom.

(b) Cytobrush and Spatula Protocol

1. Introduce an extended-tip spatula into the endocervix. Rotate the spatula through 360° pivoting at the os. Withdraw the spatula and put aside temporarily, keeping the material on the spatula.
2. Introduce an endocervical brush device into the cervix. Rotate the brush 90° - 180°. (A brush is not recommended in pregnancy, cervical stenosis, or other clinical conditions indicated by the manufacturer).
3. Spread the spatula specimen onto the left side of the slide and fix while covering the right side (see Figure 5).
4. Roll the cytobrush sample over of the right side of the slide and fix.

   Figure 5. Spread the spatula sample over the left side of the slide and fix while covering the right side. Roll the brush over the right side of the slide and fix.

5. Quality Indicators

(A) Inadequate Smears

• A satisfactory smear should show well-preserved and well-visualised squamous cells covering at least one-third of the area of a regular glass slide surface. Whereas for a liquid-based ThinPrep preparation, a minimum of 5,000 well-visualized/preserved squamous cells are needed for a specimen to be considered satisfactory.
• If fewer than these are seen because of paucity of cells, poor fixation, air-drying artefact, thick smearing, or covering of blood, inflammatory exudate (with >75% of cells obscured) or other contaminants, the smear is consider unsatisfactory.
• A smear comprising mainly endocervical cells is also considered unsatisfactory, unless the smear was intended to specifically evaluate the endocervical canal.

(B) Endocervical Cell / Transformation Zone (EC/TZ) Component

• At least 10 well preserved endocervical columnar cells or squamous metaplastic cells qualify as an EC/TZ component.
• If <10 cells are seen, the EC/TZ component is reported as absent.
• In the presence of atrophy, only definite squamous metaplastic or endocervical cells count as an EC/TZ component.

Non-Gynaecological Cytology - Smear, Fluid and Fine Needle Aspiration (FNA)

1. General Instructions

A. Smear Preparation

1. Label all slides with patient’s NRIC and initials on the frosted end of the slides using a hard lead pencil prior to smear preparation.
2. If more than one site is sampled, it is mandatory that the source of the specimen be clearly indicated on each slide.
3. Submit a maximum of 4 slides of material from any source that can be evaluated cytologically.
4. Fix slides immediately (within seconds) with cytology spray fixative (see figure 6) before air-drying occurs or immersed in 95% ethanol for at least 20 minutes.
5. Allow fixative to dry thoroughly before packaging slides in the appropriate slide container for transport.
6. If air-dried smears (for indications, see specific sites), one to be submitted together with wet fixed smears; the slides should be labelled ‘wet-fixed’ or ‘air-dried’ as appropriate.

B. Fluid

1. Submit 20 to 50 mL of fluid to the Cytology Lab within 1 hour of collection. If delay is anticipated, refrigerate at 4ºC. Exceptions include CSF and urine, which degenerate within one hour, even with refrigeration. These should be sent immediately to the laboratory.
2. Place fluid in a tightly capped, appropriately labelled container.
3. If more than one site is sampled, the source of the specimen must be indicated on each specimen container.
4. Please DO NOT submit fluid specimens in huge containers (e.g. 5L bottle, etc) or drip bags.

2. The Request Form

All specimens should be accompanied by the appropriate request form that includes:

• Patient’s name, age, sex and identification number
• Patient’s exact location (hospital/ department/ clinic/ ward / bed)
• Submitting physician’s name, MCR number and telephone number
• Time and date of collection
• Source of specimen/ anatomic site sampled
• The number and type of specimens submitted (containers of fluid, slides, etc.)
• Test requested
• Relevant clinical findings and history, including operative findings, previous biopsy number or date of previous operation
• Provisional diagnosis

Failure to provide ALL the above information will delay turnaround time for the Cytology report.
When more than one specimen is sent from the same patient at the same operation, use only one form.

Figure 6. Fixation of the Spread Sample Using a Pump Spray

Click here to view all Gynaecological Cytology (Pap smear) tests
Click here to view all Non-gynaecological Cytology tests