Cytology. Papanicolaou Smear

Transcription

1 Cytology Cytologic Studies: Papanicolaou smear (Pap test, GYN cytology), sputum cytology, fluid cytology, fine needle biopsy. Cytologic samples are cellular suspensions, which are spread, smeared or centrifuged on to glass microscope slides. These are immediately fixed with 95 % ethanol or air-dried and subsequently stained for morphologic evaluation. The optimal sample is a preparation consisting of a monolayer of viable cells with immediate (rapid) fixation. Specimens, which are routinely processed for cytology include: cervical and endocervical smears of the gynecologic tract (Pap smears); fine needle biopsies of all body sites; cerebrospinal fluids; pleural effusions; peritoneal effusions; peritoneal washes; urines; and scrapings or brushing from either skin, respiratory or gastrointestinal sites. Classically cytologic samples are divided into those that are exfoliative cytology and aspiration cytology. Papanicolaou Smear Purpose The purpose of obtaining a Pap test (a scraping of cells from the uterine cervix) is to detect cervical cancer and its precursors. Inflammation, reparative changes and other abnormalities of the reproductive tract may also be detected during the examination of the specimen. Patient Preparation Obtaining the specimen for Pap smear is usually done during routine or diagnostic pelvic examinations. Any individual preparing for medical examination may experience anxiety over the possibility of an abnormal finding being discovered. Women who are undergoing Pap test examination should be treated in a courteous, professional and efficient manner in an attempt to dispel anxiety. Concerned health care providers should always freely explain procedures and answer questions as an important in the part of the initial assessment and preparation of a patient undergoing Pap smear examination. Patients should be tested approximately two weeks after the first day of the last menstrual period. Obtaining Pap tests during menstruation usually renders the specimen inadequate for evaluation. Vaginal medications, douches, or vaginal contraceptives are discouraged for 48 hours prior to the appointment. Filling Out the Requisition The Gynecologic Cytology Requisition form complies with all government regulations. It serves as the gynecologic cytology work card, as well as the clinical requisition. This form must be completed and contain the necessary identifying and clinical items of information in order that the laboratory accepts and fully evaluates the specimen. 1. Patient Name (Last, First, Middle initial, if available) 2. Telephone number, if available 3. Medical Record Number 4. Patient location 5. Social Security Number (other identifier for outpatients) 6. Patient Date of Birth 7. Sex 8. Ordering Physician Name 9. Any additional physicians to who report should be sent 10. Billing Instructions a. Client bill b. Fee for service c. Bill insurance, Medicare or patient account 11. Diagnosis Code (required for all Medicare patients) 12. Advanced Beneficiary Notice (must be signed and submitted in certain cases for Medicare see Section Two on Compliance and use of the ABN for complete instructions)

2 13. Date and Time of Collection 14. Specimen Information (if applicable) 15. Requested Tests(s) 16. Clinical information a. The date the specimen is obtained. b. Specimen source (either cervical, vaginal, endocervical, or a combination of all three). c. The last menstrual period MUST be included. d. The patient history items should be checked or written in. e. Type of Pap test requested (conventional vs. liquid) 17. The following historical items a. Prenatal. b. Premenopausal. c. Postpartum. d. Postmenopausal. e. Previous biopsy specimen. f. Previous inadequate cytology. g. Previous abnormal cytology. h. Previous hormonal usage. i. Presence of abnormal bleeding. j. Presence of radiation therapy. k. Hysterectomy. l. IUD use. m. Other (free text). 18. One of the indications must be checked: a. Screening. b. High risk-risk factors for cervical cancer (defined as early age for sexual intercourse, multiple sexual partners, and a history of sexually transmitted disease). Risk factors for endometrial carcinoma (defined as postmenopausal women with a history of obesity, diabetes mellitus, and history of infertility). c. Diagnostic (known or suspected abnormality) Obtaining the Specimen All materials used to obtain the Papanicolaou test should be ready prior to the collection of the sample. -Supplies include: Cervical spatula, endocervical brush, a glass slide or pap vial with collection fluid, spray fixative, and slide holder. The glass slide or vial should be labeled with the patient s name and date before obtaining the specimen. The vaginal speculum should be placed using only water for lubrication (jellies can obscure the cytologic test). If a woman is over 40 years old, a vaginal pool specimen should be obtained with the blunt end of the spatula and handed to the assistant. In all women, the ectocervical sample should be obtained by firmly rotating the spatula 360 degrees against the visible portion of the cervix. The mucus plug should be removed from the endocervical canal prior to sampling with the endocervical brush. The endocervical brush should be inserted into the endocervical canal and rotated 180 degrees. (Caution: The endocervical brush should always be visible within the endocervical canal.) Preparing the Sample There are two acceptable methods for preparing a Pap test. The goal is to prepare the sample as quickly as possible. If making a smear, an even distribution of cellular material should be attempted and fix the specimen immediately. Method A: Conventional 1. Obtain the endocervical brush specimen. 2. Cover one-half of the glass slide with the paper towel or a piece of paper.

3 3. Smear the brush over the uncovered area evenly. 4. Immediately spray fix the smeared sample keeping one-half of the glass slide covered, holding the spray fixative 10 inches from the slide. 5. Uncover the remainder of the slide. Smear the ectocervical sample on the unfixed portion (avoiding the frosted end). 6. Immediately spray the remainder of the slide. Method B: Conventional 1. Obtain cervical brush specimen. 2. Either hand the brush to an assistant or place the brush on the glass slide near (not on) the frosted end. Do not smear. 3. Obtain the ectocervical sample. 4. Spread the specimen evenly toward the end of the slide. 5. Spray fix immediately holding the spray fixative 10 inches from the slide. Method C: ThinPrep 1. Obtain the specimen as either method A or B above. 2. Instead of placing on a slide, place the brush in the Thin Prep Fixative and immediately with vigor; twirl the brush in the fluid. 3. Remove the brush and discard. 4. Label the container as you would a slide, with the patients name, date and MRN #. Slide Fixation Fixatives are agents used to prevent cell distortion and to maintain cellular detail. Improper fixation usually renders a smear uninterruptible and accurate evaluations of cell populations are precluded. 1. Fixation should be done immediately after the specimen has been spread on to the slide. 2. DO NOT WAIT until after the examination is complete. Spray fixatives are carbowax and alcohol solutions, which cover the surface of the prepared smears. The carbowax prevents air-drying. Actual fixation occurs when the specimen is processed in the laboratory. 3. When using a spray fixative, the nozzle of pump spray should be held inches from the slide. 4. Holding the spray fixative too close may blow cells off the slide or cause cellular artifacts rendering the slides less useful for diagnosis. 5. Holding the spray fixative too far from the slide may result in inadequate fixation with air-drying artifact. (The use of commercially available hair spray as fixative is a common practice in some parts of the United States. Because of the variability in ingredients and quality control, HAIR SPRAY SHOULD NOT BE USED). 6. The specimen should be allowed to dry prior to placement into mailing packages. If the slide does not dry, it will adhere to the cardboard or plastic holders in transit. 95 % Ethanol (Wet Fixation) % ethanol is an ideal cellular fixative for all cytologic smears. 2. The freshly prepared smear is immediately immersed into a Coplin Jar of 95 % ethanol. 3. Fixation occurs within minutes. Immersing the smear for a longer period of time will not injure the cells. Mailing the Slides Plastic mailers, which are disposable, are recommended. Reusable cardboard holders are considered biohazardous material. Spray-fixed smears should be inserted into the plastic mailer along with the requisition form and sent to the laboratory by mail or courier. If slides are to be mailed, they should be double-packed and mailed as HAZARDOUS material in accordance with the procedures for handling and transport of domestic diagnostic specimens and biologic agents.

4 HPV Digene Hybrid Capture II Specimen Collection and Handling The types of cervical specimens recommended for use in the hc2 HPV DNA Test are listed below. Specimens taken with other sampling devices or transported in other transport media have not been qualified for use with this assay. The hc2 HPV DNA Test s performance characteristics with other specimen types and collection devices are unknown. Cervical specimens must be collected prior to the application of acetic acid or iodine if colposcopic examination is being performed. See the HC Cervical Sampler or hc2 DNA Collection Device package insert for additional specimen collection and handling procedures. Specimens in Cytyc Preservcyt Solution Specimens collected with a broom-type collection device and placed in Cytyc PreservCyt Solution for use in making Cytyc ThinPrep Pap Test slides can be used in the hc2 HPV DNA Test. Specimens should be collected in the routine manner, and the ThinPrep Pap Test slides should be prepared according to Cytyc instructions. There must be at least 4 ml of PreservCyt Solution remaining for the hc2 HPV DNA Test. Samples with less than 4 ml after the ThinPrep Pap Test has been prepared may contain insufficient material and could be falsely negative with the hc2 HPV DNA Test. Cervical Brushes The hc2 HPV DNA Test is designed for use with specimens collected and transported using the hc2 DNA Collection Device or HC Cervical Sampler (cervical brush and STM). Specimens may be held for up to two weeks at room temperature and shipped to the testing laboratory, after which specimens can be stored an additional week at 2-8 C. If the assay will be performed more than 3 weeks from collection, specimens can be placed at -20 C for up to three months prior to testing. A preservative has been added to the STM to retard bacterial growth and to retain the integrity of DNA. It is not intended to preserve viability of organisms or cells. The hc2 DNA Collection Device or the HC Cervical Sampler should not be used for collection of specimens from pregnant women. Specimens may be shipped without refrigeration to a testing laboratory; however, specimens should be shipped in an insulated container using either an overnight or 2-day delivery vendor. Cervical Biopsies Freshly collected cervical biopsies, 2-5 mm in cross-section, may also be analyzed with the hc2 HPV DNA Test. The biopsy specimen must be placed immediately into 1.0 ml of STM and stored frozen at -20 C. Biopsy specimens may be shipped at 2-30 C for overnight delivery to the testing laboratory and stored at -20 C until processed. Biopsies less than 2 mm in diameter should not be used. Non-Gynecologic Cytology Non-gynecologic cytology is the morphologic evaluation of fine needle biopsies, body cavity fluids, washings, scrapings, brushings, and urine specimens. The primary goal of non-gynecologic cytology examination is to detect malignancy. The non-gynecologic requisition form must be filled out entirely. Absence of information will limit the ability of the cytologist to fully evaluate the specimen. Sputum Cytology 1. Early morning deep cough specimens are the most cellular and most representative of the respiratory regions. 2. The oral cavity should be rinsed thoroughly prior to collection of the specimen.

5 3. A preservative agent should be added immediately, if a fresh specimen cannot be taken to the Cytology Laboratory. 4. An adequate sputum evaluation for cancer consists of three consecutive early morning samples. A single post-bronchoscopy deep cough specimen may be of great diagnostic value. 5. NOTE: Preservative agents (for home collection or accumulated specimens) may be obtained by calling the Cytology Laboratory at Breast Secretions 1. Nipple discharge or breast secretions can be obtained to evaluate the presence or absence of malignant diseases of the breast. 2. Ten frosted slides labeled with the patient s name and hospital number should be available. 3. A Coplin jar of 95 % ethanol should be used for fixation. SPRAY FIXATION SHOULD NOT BE USED. 4. Patient preparation: The nipple should be cleansed with an alcohol sponge. The breast should be stripped and the initial breast secretions discarded. [Lactiferous sinuses (near the nipple)] often hold secretions for a long period of time and contain degenerated cells. 5. Initial expressions from the nipple should always be discarded prior to making smears.] 6. After the initial secretions are discarded, allow a drop to accumulate. 7. Support the areola and nipple with one hand, and with the other hand place a slide upon the nipple, pause to allow material to spread laterally, and then draw the slide quickly across the nipple. 8. Immediately drop the slide in the bottle of 95 % ethanol fixative. Do not air dry. 9. The procedure is repeated using the ten slides. 10. It is important to make this many slides because when carcinoma is present, the last slides in the series are usually diagnostic. CSF 1. The optimal volume for CSF fluid for the detection of malignancy is 10 ml. Once fluid is drawn, prepare for transport in a sterile, threaded tube for transport. 2. No preservatives should be added, but if transport is delayed, the specimen must be placed on ice to avoid degradation. 3. Shunt collections should be noted on the requisition. 4. If lymphoma/leukemia is a clinical consideration half of the specimen should be aliquoted at bedside for flow cytometric analysis. 5. Fluid volumes of 1 ml or less are inadequate for the consideration of malignancy shedding into CSF space. Surface Scraping Cytology (Skin or Mucosal Scrapings) Cytologic samples of these slides are taken to rule out the possibility of malignancy or infection when biopsy of the area is not desirable. 1. Preparations: Glass slides with frosted ends, labeled with the patient s name and hospital number. Other supplies include: 95 % ethanol fixative in a Coplin jar, wooden spatula, or endocervical brush, and glove with gauze. 2. Sampling: A moistened gauze square of single thickness over gloved finger or endocervical brush or a moistened wooden spatula should be used for sampling. (moistening the spatula is essential to avoid airdrying.) Endocervical brushes may be most useful for mucosal lesions. Keratotic lesions should be abraded to remove much of the surface keratin. Stop abrading if small bleeding points appear. Using fresh gauze, brush or spatula to sample, smear the specimen on the slide. Vesicular lesions should be ruptured and the base sampled (vesicle fluid is not acceptable due to degeneration). Red lesions are sampled without special preparations. 3. The specimen is immediately smeared on the glass slide using a single stroke. It is then rapidly fixed by plunging into 95 % ethanol. Spray fixatives SHOULD NOT be used due to rapid air-drying of these samples.)

6 Major Body Cavity Fluids Effusions These specimens are the result of fluid accumulation in the potential spaces of the pleura, peritoneum, or pericardium. 1. Activation of the coagulation system with fibrin clots must be avoided as well as cellular degeneration. 2. In order to avoid coagulation, heparin should be added to the collection bottles prior to the addition of the specimen. (Three units per one milliliter of fluid to be added.) 3. Once the effusion fluid has been added to the collection bottle, mix the heparin and the fluid by agitation. 4. Immediately deliver the specimen to the Cytology lab to avoid degeneration. 5. If this is not possible, an effusion specimen may be safely refrigerated overnight, but no longer than 24 hours. 6. Degeneration may occur in vivo and re-sampling may be essential. Specimens submitted for cytology are NOT suitable for microbiologic analysis or culture. 7. A separate sample should be submitted to Microbiology with the appropriate requisition, as well as to the Hematology Laboratory for cell count and differential, if desired. Minor Body Cavity Fluids 1. The minor body cavity fluids include cerebrospinal fluids, eye chamber fluids, joint-space fluids, peritoneal cul-de-sac fluids and cyst fluids. 2. These fluids are usually less cellular and of smaller volumes and rarely present a problem with clotting. Because of their low cellularity and relative difficulty in repeat sampling, proper specimen handling is essential. 3. These specimens should all be collected using sterile technique. The clinician should not attempt to make smears for cytologic examination. Rapid transportation to the laboratory is essential. Any delay in processing will affect morphology adversely. Urine 1. The patient should be well hydrated prior to the collection of the specimen, if possible. Patient ambulation is favorable for increasing the natural exfoliation of cells into the urine. 2. Once 20 to 50 milliliters of urine is collected, it must be placed on ice and brought to the laboratory as soon as possible. 3. First morning urines are NOT suitable for cytologic diagnosis. 4. If catheterization is unavoidable, it must be clearly noted on the requisition as catheterization may cause cellular changes, which can be mistaken for low-grade papillary carcinomas of the bladder. 5. Do NOT collect a specimen after intravenous pyelography. 6. Urinalysis and microbiological evaluation of the urine are not performed in cytology and a separate specimen should be sent to chemistry or microbiology for these studies. Brushing and Washing Specimens These specimens are obtained by washing cells from surfaces or abrading surfaces with small brushes. Classically, this involves the gastrointestinal and respiratory tracts and peritoneal cavity. In general, all samples must be processed using balanced salt solution; washings are generally attained after brushing. All samples should be transported rapidly to the laboratory. Brushes should be immediately submerged in 10 to 16 milliliters of balanced salt solution in a sterile container. The brush should be agitated briskly in the fluid and clipped off into the container. (Please leave two inches of cable in the brush.) All specimens must be labeled as to the specific site, especially if several areas are sampled. Respiratory tracts brushing and washings 1. These specimens are usually acquired via fiber optic bronchoscopy. 2. The brush must be removed through the scope with retraction into the protective sheath. 3. The brush should be submitted in a Cytolyte cytology fixative, leaving at least one inch of cable above the brush. 4. Brushing specimens should be obtained prior to cutting (forceps) biopsies and followed by saline wash. 5. These should be submitted fresh to the laboratory as soon as possible.

7 Gastrointestinal tract 1. Gastric and esophageal brushes and washes are the most common type of gastrointestinal tract specimen. 2. Both are acquired by endoscopy, however, washings may sometimes be obtained by nasogastric tube. 3. Any washing from the esophagus or gastric area must be placed on ice and delivered immediately to the laboratory to prevent degeneration, which occurs due to the natural acidity of gastric contents. 4. Esophageal and gastric brushing should be placed in a Cytolyte cytology fixative and sent to the laboratory. 5. Colonic and intestinal samples must be preceded by meticulous colon preparation. Peritoneal washings 1. These samples are usually used for staging of abdominal neoplastic disease, especially of gynelogic origin. 2. After washing with a balanced salt solution or saline, the specimen should be submitted immediately to the cytology laboratory for processing. Urinary bladder 1. Urinary bladder washings or arbitrage are recommended for morphologic evaluation in suspected cases of transitional cell carcinoma. 2. Because degenerative changes may occur, such washings should be submitted immediately to the cytology laboratory. Fine Needle Biopsy The fine needle aspiration biopsy is a rapid, inexpensive, simple cytologic technique that can be done on an outpatient basis. Both palpable subcutaneous masses and deep-seated lesions of the head, chest or abdomen can be sampled radiologically. A cytotechnologist assists the physician performing the fine needle (if unavailable in an office situation, cytotechnologists are available to train your office personnel for preparatory techniques). A pathologist is available to perform FN of palpable masses. Please call or to schedule an appointment. The technique employs the following equipment: 1. several 10 ml disposable plastic syringes, or 25 gauge needles 3. an FN handle 4. alcohol swabs 5. a Coplin jar containing 95 % ethanol clean glass microscope slides. 7. Local anesthesia (1 % solution of Xylocaine, Lidocaine or other anesthesia) is used for skin infiltration. Physiologic saline solutions or a small vial of fixative may be used for needle rinses. Obtaining a palpable fine needle aspirate: 1. The skin is cleansed using an alcohol swab. 2. A small amount of local anesthetic is infiltrated into the skin and subcutaneous tissue. There are two accepted methods for obtaining fine needle cytologic specimens: the aspirate technique and the no-aspirate technique. To perform an aspiration gauge needle attached to a syringe is inserted into a lesion.

8 2. Negative pressure is applied and the needle moved back and forth within the lesion, with a slight redirection of the needle during each motion. 3. Needle pressure is released prior to removing the needle from the skin. 4. The needle is withdrawn. 5. The syringe and needle are handed to the cytotechnologist who expresses the cells and smears them between two slides 6. A single slide is air-dried and the other is immediately plunged into the Coplin jar of 95 % ethanol. 7. Needles may be rinsed with a saline solution or fixative for cell block preparation 8. This is repeated four to six times until the lesion is well -sampled. No aspirates may also be performed if preferred by the operator. A no aspirate technique is simply: 1. a 25-gauge needle without a syringe placed within the lesion after patient preparation and anesthesia. 2. Without covering the hub, the needle is moved back and forth sharply, redirecting the tip with each movement. 3. The needle is withdrawn and the contents expelled on to a slide and processed in a similar manner as an aspirate technique. 4. Four to six needle punctures are performed until the lesion is well sampled. For further questions regarding technique, scheduling fine needle biopsies, or requesting cytotechnologist assistance, please call or