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Appendix F

CytologySpecimen Collection and Processing

Gynecological Cytology Specimens (Pap smear)

ThinPrep Pap test

Principle

A gynecological cytology specimen (Pap smear)is an evaluation for the presence of abnormalcells, which may be indicative of malignancy orother conditions requiring treatment. It isimportant to sample the cervix or vagina wellwith minimal artifact and obscuring materials.

Precautions

1. Gloves should be worn when collectingand handling the specimen.

2. Specimens should be taken beforepelvic examination.

3. The patient should not douche or usevaginal medication for 24 hours beforethe specimen is obtained. This shouldnot, however, prevent obtaining aspecimen. Inform the patient that thetest may be unsatisfactory so that shewill not unduly be alarmed if a repeatPAP is later required.

4. Do not use lubricant. If necessary, thespeculum may be moistened withnormal saline. Avoid using water, whichis hypotonic and will produce cellulardistortion.

5. Avoid, if possible, taking specimenduring normal menses. However, ifthere is abnormal bleeding, obtainroutine PAP and consider directendometrial specimen.

Materials

1. Cytyc Preservcyt solution vial.Preservcyt solution is a methanol-basedbuffered preservative solution. Storethe vials at 15º – 30º C. (59º – 86º F.)

2. Plastic spatula3. Endocervical brush4. GYN cytology requisition form number

245 and Client Request Form.

These supplies can be obtained from the Scott &White Reference Lab:

PreserCyt Solution Medescand Cytobrush Plus GT

(endocervical brush & plastic spatula)

NOTE: Cotton tip swabs and wooden spatulasshould not be used to obtain specimens forThinPrep PAP test.

Collecting the Specimen

1. Write the patient’s name and medicalrecord number on the vial or place thepatient’s identification label on the vial.This is essential to prevent a mix-up ofthe specimens during processing.

2. Expose cervix with the speculum. Thecervical surface should not be wiped;wiping it will remove the cell-richadherent cervical mucus.

Cervical Scrape

Scrape the external os 360º with the plasticspatula and as quickly as possible place thespatula into the preservcyt solution vial, swirlingthe spatula vigorously in the vial 10 times.Never induce bleeding by scraping the cervixvigorously.

Endocervical Brush

Insert the brush into the cervical os and rotategently. It is recommended that the brush berotated only 180º. More rotation may causeexcessive bleeding. Rinse the brush as quicklyas possible in the preservcyt solution vial byrotating the device in the solution 10 times whilepushing against the preservcyt vial wall. Swirlthe brush vigorously to further release material.For thick mucoid specimens collected using thebrush, to further release endocervical cells thatmight be entrapped in mucous; use the concaveside of the spatula and scrape down the brushbristles a few times and on different sides of thebrush. This can be done while holding the brushin the vial with the left hand and using thespatula to scrape with the right hand.

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Appendix F: Cytology continued

Vaginal Scrape:

For specifically desired hormonal evaluation(maturation index), gently scrape lateral wall ofupper third of vagina. Rinse the spatula asquickly as possible in the preservcyt solution vialby swirling the spatula vigorously in the vial 10times.

1. Tighten the cap so that the line on thecap and the line on the vial meet.

2. For best results, please follow thesepreparation steps diligently.

Method for Submitting Specimen to theLaboratoryClients will submit a request form and a form245 with the patient's name, medical recordnumber, and the appropriate clinical andbilling information. This included properdiagnostic codes, specimen source and otherclinical data such as last menstrual period(LMP), previous treatment, previousabnormals, colposcopic findings, hormonalstatus, etc. Fill in the date and time thespecimen is obtained. The specimen vialshould be placed in a biohazard bag and thecompleted requisition form placed in the sidepocket of the bag. Specimens collected after5:00 PM, on weekends or holidays should beheld until the following workday.

Non-Gynecological Cytology Specimens

Principle

It is important that high quality diagnostic materialis provided for cytopathologic examination.

Precautions:

Gloves should be worn at all times when handlingunfixed specimens in accordance with theDepartment of Pathology Bloodborne PathogenPolicy. All NON-Gyn cytology specimens mustbe handled using face protection.

Procedure for Specimen Collection

All specimen containers and slides should beproperly identified and labeled with the patient’sname and medical record number.

A completed requisition form that matches thespecimen identification should be submitted withthe specimen. The form should bear patientidentification data, date and time of collection,physician/resident name, source of specimen andpertinent clinical information.

Failure to properly identify specimens, ormismatches between specimens and requisitionforms will result in delay of processing orrejection of the specimen.

LUNG

Sputum

The patient must rinse his/her mouth with water,bend horizontally to the waist and press hishands against the abdomen (just below ribtraction of diaphragm) and expectorate directlyinto the container.

Sputum specimens are collected in the freshstate in sputum cups and transported to thelaboratory inside biohazard plastic bags. Therequisition form containing the demographic data,clinical information, date and time of collectionmust be attached. Specimens received duringthe night and weekends are to be placed in therefrigerator and delivered to cytology the nextworking day. Specimens not processed within 18hours should be fixed with 50% to 70% alcohol.

Bronchial Washing

The bronchial was specimens are collected in thefresh state in a container and transported in abiohazard plastic bag. The completed requisitionform must be attached. Reference to the specificsite of washing should be included (e.g. rightupper lobe). Requests for special studies (e.g.GMS stains, flow cytometry, etc.) should beindicated.

Bronchial Brush

The smear is made at the time of endoscopy byrolling the brush on a totally frosted slide or slideswhich are immediately immersed in a Coplin jarwith 95% alcohol.

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Appendix F: Cytology continued

FLUID FROM SEROUS CAVITY

All effusions submitted for cytologic evaluationmust be heparinized at the time of collection toprevent coagulation. The recommended quantityof heparin (1:10,000) is 1 ml heparin to 300 mlbody cavity fluid. If the patient has been bedridden, it is advisable to gently rotate him prior totapping the fluid filled area; this is necessary tore-suspend those cells, which have settled withinthe body cavity due to their heavy cellulardensity.

The specimen should be brought fresh to thecytology lab immediately following the procedureduring the day. Should the procedure need to beperformed at night or on weekends, the fluidshould be placed in a refrigerator. Pleur-evaccontainers must not be submitted to the cytologylab.

CEREBROSPINAL FLUIDS

Spinal fluid for cytologic examination obtainedduring working hours (M-F from 5:00 AM to 6:00PM) should be immediately delivered to thecytology lab to be processed. Specimensobtained after 6:00 PM or on weekends/holidaysshould be mixed with an equal volume of 50% or70% ethyl alcohol and placed in the refrigerator inthe microbiology lab to be delivered the followingworking day.

URINE

All voided specimens should be collected as amid-stream clean catch. The first morningspecimen should be discarded.

To the urine specimen, add an equal part of 50 to70% alcohol. The patient’s name should beplaced on the urine container. The clinical datashould include whether the urine is voided orinstrumented. The presence or absence of aprevious tumor or previous treatment should alsobe noted and the cytoscopy findings indicated. Aform that included the requested clinical data hasbeen attached to this page and can beduplicated. It should be attached to all requisitionforms of urine specimens.

ENDOSCOPIC BRUSHINGS

Gastric, esophageal, duodenal, bile duct orcolonic brushings are done on completely frostedslides. The smears are prepared quickly andplaced immediately in 95% ethyl alcohol. Air-drying should be avoided.

FINE NEEDLE ASPIRATION

Direct smears made at the time of aspiration areimmersed immediately in Carnoy’s fixative(provided by the cytology lab) for approximately 5minutes then transferred into 95% ethanol.Caution should be taken not to leave the slides inthe Carnoy’s fixative for more than 5 minutes,because this will result in cellular distortion andartifacts. The purpose of this short immersion inCarnoy’s fixative is to lyse the red blood cells andprevent obscuring of cellular detail by blood.

Specimens that are not directly smeared, are tobe collected in 20 ml tube of a 1:1 solution of50% ethyl alcohol and Ringer’s solution (5 mleach). The sample is flushed into the containerand sent with the appropriate cytology form to thelab for processing. Aspirates from cystic lesionscan be forwarded to the lab in the syringe (afterdiscarding the needle) if they are sentimmediately after the procedure is completed. Ifthat is not possible, they should be flushed intothe above-mentioned fixative solutions.

BREAST FLUID SECRETION

The area of greatest accumulation of secretion isfound immediately below the nipple and theareolar area. A breast pump may be used,although material collected for cytologicevaluation is more frequently obtained throughspontaneous secretions. Care should be takennot to manipulate the breast unnecessarily. Thematerial for cytologic evaluation is collected byplacing a frosted-tip slide against the nipple andsmearing the fluid quickly over the slide.Immediate fixation may be accomplished if thepatient (or an assistant) is allowed to hold theopen bottle of 95% ethanol in front of the breastso that the slide can be immediately dropped intothe fixative. Air-drying should be avoided as itmay render the specimen non-diagnostic.