Made with Xara © Arkansas Pathology Associates 2010 Gynecologic Cytology Specimens Patient Preparation To optimize collection conditions, a woman should:  1.  Schedule an appointment approximately two weeks (10-18 days) after the first day of her last menstrual period. 2.  Not douche 48 hours prior to the test. 3.  Not use tampons, birth control foams, jellies, or other vaginal creams or vaginal medications for 48 hours prior to the test. 4.  Refrain from intercourse 48 hours prior to the test. Test Requisition Under the supervision and guidance of the physician, a laboratory requisition must be legibly and accurately filled out before obtaining the cellular sample.  The laboratory requisition is the main communication link between the physician and the laboratory.  The requisition form should have the following information as required by CLIA ‘88: 1.  Patient’s name (any name change in the past 5 years should be noted) 2.  Age and/or date of birth 3.  Menstrual status (LMP, hysterectomy, pregnant, postpartum, hormone therapy) 4.  Previous Pap history, abnormal cervical cytology results, previous treatment, biopsy or surgical procedure and results 5.  Source of specimen (e.g., cervical, vaginal) Appropriate clinical history provided by the physician on the requisition should include: 1.  Hormone/contraceptive use 2.  Relevant clinical findings (abnormal bleeding, grossly visible lesion, etc.) Patient’s SSN, physician and health facility names, insurance information and medicare information should be provided where applicable. Labeling the Sample Write the patient’s name and another patient identifier on the PreservCyt Solution vial.  Examples of acceptable identifiers include, but are not limited to, patient’s date of birth or SSN, or tracking or requisition number.  A location (e.g., hospital room) is not an acceptable identifier. Visualization of the Cervix for Collection of an Adequate Sample 1.  Collection of a cervical cytology specimen is usually performed with the patient in the dorsolithotomy position. 2.  A sterile, or single-use bivalve speculum of appropriate size is inserted into the vagina without lubrication.  Warm water may be used to facilitate insertion of the speculum.  The position of the speculum should allow for complete visualization of the os and ectocervix.  If a lubricant must be used due to patient discomfort or other circumstances, it should be applied sparingly on the outer portion of the speculum with great care to avoid the tip.  Hologic (ThinPrep) has evaluated a variety of popular lubricants and found that those containing an ingredient known as “carbomers” or “carbopol polymers” are prone to interfere with liquid based Pap tests. 3.  The transformation zone is the site of origin for most cervical neoplasia and should be the focus of cytology specimen collection.  The transformation zone may be easily visualized or may be high in the endocervical canal.  Location varies not only from patient to patient, but in an individual over time.  Factors producing variation include changes in vaginal pH and hormonal changes including pregnancy, childbirth, menopausal status, and hormonal therapy. 4.  In postmenopausal patients or women who have received radiation therapy, cervical stenosis may prevent visualization of the transformation zone.  It remains important to sample the endocervix in these patients.  This may require more extensive clinical procedures. 5.  If a patient has had a hysterectomy, a vaginal sample is sufficient, with particular attention to sampling the vaginal cuff.  ThinPrep Pap Test Specimen Collection, Adequacy, Requisition, and Transportation Techniques for Sample Collection Spatula and Endocervical Brush Technique 1.  The ectocervix should be sampled before the endocervix/transformation zone.  First, a sample of the ectocervix is taken using a plastic spatula.  The notched end of the spatula that corresponds to the contour of the cervix is rotated 360° around the circumference of the cervical os. 2.  The spatula is rinsed in the PreservCyt Solution vial by swirling the spatula vigorously in the vial 10 times. 3.  Sampling of the endocervix requires insertion of the endocervical brush into the endocervical canal until the bristles closest to the hand are visible.  The brush is rotated 45-90° and removed. 4.  The brush is rinsed in the PreservCyt Solution vial by rotating the device in the solution 10 times, while pushing against the PreservCyt vial wall.  If material still remains on the brush, take the spatula and scrape the material from the brush while holding it in the PreservCyt Solution vial.  Discard the brush and spatula. 5.  The vial cap is tightened so that the torque line on the cap passes the torque line on the vial. Broom-Like Device Technique 1.  The ectocervix and endocervix are collected simultaneously.  The central bristles of the broom are inserted into the endocervical canal until the lateral bristles bend fully against the ectocervix.  The sampling device is rotated 360° in the same direction five (5) times while maintaining gentle pressure. 2.  The broom is removed and rinsed in the PreservCyt Solution vial by pushing the broom into the bottom of the vial 10 times, forcing the bristles apart.  As a final step, the broom is vigorously swirled to further release material.  Discard the device. 3.  The vial cap is tightened so that the torque line on the cap passes the torque line on the vial. Transporting Specimen 1.  The vial is placed in the specimen transport bag and sealed.  The requisition is placed in the pouch outside the sealed bag. 2.  The specimens are transported to the cytology laboratory by courier.  If the specimens are to be mailed, contact the cytology laboratory for instructions. Conventional Pap Smear Specimen Collection, Adequacy, Requisition, and Transportation Techniques for Sample Collection Spatula and Endocervical Brush Technique 1.  The ectocervix should be sampled before the endocervix/transformation zone.  First, a sample of the ectocervix is taken using a plastic (or wooden) spatula.  The notched end of the spatula that corresponds to the contour of the cervix is rotated 360° around the circumference of the cervical os. 2.  The sample on the spatula is spread evenly and thinly lengthwise down one half of the labeled slide surface, using a single uniform motion.  Immediately spray with fixative.  Follow the manufacturer’s instructions on the container and package insert.  Generally, spray fixatives should be 6-10 inches from the glass slide when applied. 3.  Sampling of the endocervix requires insertion of the endocervical brush into the endocervical canal until the bristles closest to the hand are visible.  The brush is rotated 45-90° and removed. 4.  The endocervical brush is then rolled along the remaining half of the labeled slide surface by turning the brush handle and slightly bending the bristles with gentle pressure.  Immediately spray with fixative. Broom-like Device Technique 1.  The ectocervix and endocervix are collected simultaneously.  The central bristles of the broom are inserted into the endocervical canal until the lateral bristles bend fully against the ectocervix.  The sampling device is rotated 360°  in the same direction five (5) times while maintaining gentle pressure. 2.  The broom is removed and with a single paint stroke motion the cellular sample is transferred down the long axis of the labeled surface of the slide.  The broom is turned over and the paint stroke motion is repeated over the same area.  Immediately spray with fixative.  Follow the manufacturer’s instructions on the container and package insert.  Generally, spray fixatives should be 6-10 inches from the glass slide when applied. Transporting Specimen 1.  Place slide(s), when dry, in slide holder. 2.  Place slide holder with the slide and completed requisition form in a specimen transport bag. 3.  If the specimen is not transported by courier, but by mail, request a special mailer from the cytology laboratory. The Bethesda System 2001 Gynecologic Cytology Classification Specimen Adequacy Satisfactory:  Satisfactory for evaluation, but may include any quality indicators, e.g., absence of endocervical component, partially obscuring blood, inflammation, etc. Unsatisfactory:  Unsatisfactory for evaluation of epithelial abnormality because of reason specified and should be repeated. Descriptive Interpretation Negative for Intraepithelial Lesion or Malignancy:  Negative for squamous cell abnormalities.  Organisms and other non-neoplastic findings are included under this category. Organisms: -Trichomonas vaginalis -Fungal organisms morphologically consistent with Candida spp. -Bacteria morphologically consistent with Actinomyces spp. -Cellular changes consistent with Herpes simplex virus Other Non-Neoplastic Findings: -Reactive changes -Radiation changes -Parakeratosis and/or hyperkeratosis -Atrophy -Endometrial cells present in a woman 40 years and older Epithelial Cell Abnormalities Squamous Cell -Atypical squamous cells      -of undetermined significance (ASC-US)      -cannot exclude HSIL (ASC-H) -Low grade squamous intraepithelial lesion (LSIL)  encompassing:  HPV/mild dysplasia/CIN I -High grade squamous intraepithelial lesion (HSIL)  encompassing:  moderate and severe dysplasia, CIS/CIN 2 and CIN 3      -with features suspicious for invasion (if invasion is suspected) -Squamous cell carcinoma Glandular Cell -Atypical      -endocervical cells (NOS or specify in comments)      -endometrial cells (NOS or specify in comments)      -glandular cells (NOS or specify in comments) -Atypical      -endocervical cells, favor neoplastic      -endometrial cells, favor neoplastic      -glandular cells, favor neoplastic -Endocervical adenocarcinoma in situ -Adenocarcinoma      -endocervical      -endometrial      -extrauterine      -not otherwise specified (NOS) Other Malignant Neoplasms (specify) Maturation Index The Maturation Index (MI) expresses the relationship of parabasal to intermediate to superficial squamous cells.  The MI will be reported as relative percentages of these cells and written as a ratio:  parabasals %:  intermediates %:  superficials %.  The response of the squamous epithelium of the vagina to various hormonal stimuli can show great variations from patient to patient.  The only two absolute cell patterns are (1) a predominance of superficial cells that indicates the presence of estrogen, and (2) a predominance of parabasal cells that indicates absence of estrogenic stimulation.  The Maturation Index must be taken from the lateral vaginal wall. Materials Needed 1.  Microscopic slide with frosted end.  Write the patient’s name on the label end of the slide (frosted slide) with a lead pencil.  Also, indicate on the label (V) for vaginal smear if a cervical/endocervical smear is also being collected.  ThinPrep vial:  write the patient’s name and one other identifier on the vial label.  If a cervical/endocervical sample is being collected as well, place samples in two separate vials.  Write the specimen source on each vial. 2 . Cytology spray fixative.  Use this fixative for the conventional slide method. 3.  Speculum.  Use water, not lubricant, on speculum and shake off excess. 4.  Collection device.  Wooden or plastic spatula. 5.  Gyn cytology form.  Complete the form with patient information and mark Maturation Index under Ancillary Testing. 6.  Slide holder for glass slide (conventional method). 7.  Specimen transport bag.  Place specimen in bag and seal.  Place the completed requisition form in the pouch outside the sealed bag. Specimen Collection and Preservation Vaginal Scrape 1.  Scrape the lateral wall of middle third of vagina. 2.  Conventional Method:  Spread evenly on slide and immediately spray with fixative.  Allow to dry (5-10 minutes) and place in a slide holder.  Mark slide as V (vaginal) if a cervical/endocervical smear is submitted also.  ThinPrep Method:  Place specimen in ThinPrep PreservCyt vial.  Write specimen source (vaginal) on the vial label if a cervical/endocervical specimen is submitted also. Non-Gynecologic Cytology Specimens Materials Needed: 1.  Instruments for collection 2.  APA requisition form-Complete the form with the following information:  patient’s name, age, gender, physician’s name and health facility submitting the specimen, billing information, date of collection, source of specimen, and clinical history. 3.  Container with cytology fixative-CytoLyt Solution for fluid specimens and cytology spray fixative for smear preparation specimens.  All fluid cytology specimens must be collected in cytology fixative or add the fixative shortly after collection.  Write the patient’s name, source of specimen and doctor’s name on the specimen container label. 4.  Microscopic glass slides with frosted end and slide holder for prepared slide specimens-Write the patient’s name on the frosted end of the slide with a lead pencil.  Do not use an ink pen, it washes off in the staining procedure. 5.  Specimen transport bag. Specimen Collection and Preservation Sputum When a pulmonary lesion is suspected, a complete sputum series should be examined.  This consists of a fresh morning specimen each day for three days.  A post bronchoscopy specimen may be included in the series. Method I:  Early Morning Spontaneous Deep Cough Technique 1.  Patient is given a labeled specimen collection cup containing 30 mL of CytoLyt Solution.  One cup should be provided each morning for three consecutive days.  DO NOT COLLECT THREE SPECIMENS IN ONE DAY. 2.  Caution the patient that only sputum is to be collected, not material from sinus drainage or saliva. 3.  Patient should rinse mouth with water. 4.  Instruct the patient to cough deeply several times the first hour after awakening and expectorate into the collection cup. 5.  Place lid tightly on specimen cup and shake for a few seconds. Method II:  Sputum Induction Technique If the patient is non-productive of satisfactory specimens, the induction technique should be administered.  Various aerosol instruments are available and instructions for use accompany each.  The object of the aerosolization is to introduce a significant amount of water into the lungs.  Irritants or mucolytic agents can be added. 1.  Explain the procedure to the patient. 2.  Before beginning, ask the patient to clear his throat and wash his mouth out with water. 3.  Administer the aerosol. 4.  Sputum should be expectorated into a collection cup containing 30 mL of CytoLyt Solution. 5.  Sometimes, if an adequate sample cannot be produced using an aerosol, the patient will have a productive cough within the next 24 hours.  The patient should be given a collection cup containing CytoLyt Solution and instructions for collecting a sputum sample during this period of time. Bronchial Washings and Bronchoalveolar Lavage After the specimen is collected, put the entire specimen into a collection cup of 30 mL of CytoLyt Solution.  The specimen can be sent fresh to the cytology laboratory, if it can be sent immediately after collection. Bronchial Brushing Method I Immediately after the brush is withdrawn from the bronchoscope, cut the wire a short distance from the brush and insert into the CytoLyt Solution. Method II Direct smears may be made by quickly rotating the brush gently on a glass slide labeled with the patient’s name.  Fix immediately with cytology spray fixative.  Follow the directions on the spray can.  Allow to dry (5 to 10 minutes) and place in slide container for transportation to the cytology laboratory. **Our laboratory prefers the first method rather than the slide preparation technique.** Breast Nipple Secretions Nipple secretions should be collected by applying the slide directly to the nipple and then smearing the material collected.  Immediately fix the smear with the cytology spray fixative.  Follow the directions on the spray can.  Allow to dry (5 to 10 minutes) and place in slide container for transportation to the cytology laboratory.  Gastric and Esophageal Brushing Method I Immediately after the brush is withdrawn from the instrument, cut the wire a short distance from the brush and insert into CytoLyt Solution. Method II Direct smears may be made by quickly rotating the brush gently on a glass slide labeled with the patient’s name.  Fix immediately with cytology spray fixative.  Follow the directions on the spray can.  Allow to dry (5 to 10 minutes) and place in slide container for transportation to the cytology laboratory. **Our laboratory prefers the first method rather than the slide preparation technique.** Gastric and Esophageal Washings After the specimen is collected, put the specimen for cytologic exam into a collection cup of CytoLyt Solution.  The specimen can be sent fresh to the cytology laboratory, if it can be sent immediately after collection. Body Cavity Fluids, Cerebrospinal Fluid, Urine, and Other Fluids After the specimen is collected, add the specimen to an approximately equal volume of CytoLyt Solution.  Body Cavity Fluids and Other Fluids:  If the volume of specimen exceeds 30 mL, add only 30 mL of the specimen to the 30 mL of fixative in the collection cup and submit the remaining specimen unfixed.  Keep the unfixed portion of the specimen refrigerated until picked up by the courier. Urine:  Add the specimen to the 30 mL of fixative in the CytoLyt Solution collection cup.  If there is more specimen than the collection cup will hold, discard the remaining specimen. Urine for UroVysion Studies:  Collect urine specimen as specified in the above instructions.  Mark FISH orders under ancillary testing on the requisition form.  Fine Needle Aspiration Method I 1.  Have nearby a collection cup with 30 mL of CytoLyt Solution and 2 glass slides for air-dried smears. 2.  After the aspiration biopsy has been completed and the needle withdrawn, detach the needle from the syringe, fill the syringe with air, and reattach the needle.  The bevel of the needle should be placed directly on the glass slide near the label end.  Advance the plunger of the syringe to express a small drop of aspirate onto the slide.  Invert a second glass slide over the drop, and as it spreads, gently pull the two slides apart horizontally.  Let these slides air dry. 3.  Expel the remaining specimen into the collection cup of CytoLyt Solution.  Then draw the fixative into the syringe to wash out remaining specimen.  Expel into collection cup. Method II 1.  After the aspiration biopsy has been completed, the needle is detached from the syringe and air is drawn into the syringe barrel. 2.  The needle is reattached to the syringe.  The material in the needle is carefully expelled in a single drop toward the label end of a glass slide.  The open edge of the needle bevel is directed down toward the slide during expression of material. 3.  Another slide is placed face to face with slide containing specimen.  The specimen is allowed to spread without applying pressure.  If tissue fragments are present, they may be flattened with very slight pressure.  The ends are grasped and the slides are pulled apart in opposite directions.   CAUTION:  When detaching and reattaching the needle, use a needle recapping device. 4.  Let 1 or 2 slides air-dry for Diff-Quik staining and spray the remaining smears immediately with cytology spray fixative.  Follow the directions on the spray can.  Allow to dry (5 to 10 minutes) and place in slide container for transportation to the cytology laboratory. **Our laboratory prefers the first method rather than the slide preparation technique. Transportation 1.  Place specimen in a specimen transport bag and seal.  Place the completed requisition in the pouch outside the sealed bag. 2.  Transport specimens to the cytology laboratory by courier.  If specimens are to be  mailed, contact the cytology laboratory for instructions.