The primary function of the Histology/Pathology Laboratory is to evaluate specimens for the absence or presence of neoplasia; however, other conditions may also be diagnosed cytologically. Cellular preservation, important to the microscopic evaluation of cells, is dependent upon proper specimen collection.

This section describes the requirements for submitting cytological specimens. Additional help and information may be obtained by contacting the HISTOLOGY/CYTOLOGY laboratory at 423-431- 6385. Pathology client accounts may be set up by contacting the Watauga Pathology Associates office manager at 423-431-1310.

Histology/Cytology Laboratory Location and Hours of Service

Histology Technical Specialist - 423-431-6385

The Histology/Cytology Laboratory is open Monday through Friday (except holidays) from 8:00 AM. to 4:00 P.M. Specimens may be brought directly to the laboratory during these hours or delivered to the Clinical Laboratories, after hours.

Histology/Cytology Laboratory Turnaround Time

Test results for non-gynecologic specimens received in the cytopathology laboratory by 10:00 AM will generally be available by 5:00 P.M. the same day. Results for specimens received after 10:00 AM will generally be available the next workday. Test results for gynecologic specimens are available within 5 days of receipt. RUSH specimens must be clearly marked as such and are limited to the identification of microorganisms in critically ill patients or malignancy in patients whose therapy depends upon the outcome and must be initiated as soon as possible. The cytopathology staff will coordinate requests for RUSH specimens.

Test results may be obtained by calling 423-431-6389

To schedule an FNA, call 423-431-6389

Information regarding specimen-processing requirements may be obtained from the cytopathology laboratory at 423-431-6385

Histology/Cytology Test Requisitions

A Cytopathology test requisition form must accompany all cytology specimens. The patient information should be printed and the following must be included:

 Patient's last name, first name, & middle initial  Address, if an outpatient  Age/Date of Birth  Name of ATTENDING physician including first name or initial.  Medical Record Number or SS number  Location of patient

Information Clinical history/information and the exact source of the specimen mustalso be provided. For CSF and other body fluids a cytopathology requisition is still required even if the specimen is submitted through the Hematology laboratory and their form is filled in.

Cytopathology Specimen Requirements

Specimen containers, slides and test requisition must be labeled with patient's identification.

Clotted fluid specimens are not acceptable.

Specimens must be in leak-proof containers from which the specimen can be safely extracted without splattering, leaking, or the use of needles. Drainage bags, vacuum bottles, and other containers, which cannot be opened without leakage of fluids, are not acceptable and the specimen will be rejected.

For gynecologic specimens, the patient's name must appear on the slide cardboard holder or container and the test requisition form and all must be identical. Improperly labeled specimens will be rejected.

24-hour urine specimens are not acceptable due to the deleterious effects of the acid environments on cell preservation. These specimens will be discarded and a fresh specimen requested.

Causes for Sample Rejection:

 Improper labeling  Incomplete requisition, including failure to provide pertinent HISTORY. Improper specimen collection.

Collection Instructions for Various Specimen Types for Cytology:

Breast Discharge Cytoloqy: Nipple discharge. Label frosted end of slide with patient's LAST name with graphite pencil. Gently strip subareolar area and nipple with thumb and forefinger. When secretion occurs, allow pea-sized drop to accumulate on apex of nipple. Place slide on nipple and then draw slide quickly across nipple. Fix slide IMMEDIATELY with spray fixative or by immersion in 95% alcohol. Repeat procedure until discharge is depleted. If smears are prepared from both breasts, label each slide as left or right. Deliver in slide holders or jar with completed requisition to the Cytopathology Laboratory.

Bronchial Brushings: Brushing of lesion area. Break the brush off into a collection vial of CYTO- RICH. Label vial completely. Any slides prepared should be delivered in slide holders or jar with completed requisition to the Cytopathology Laboratory.

Bronchial Washings: Collect the specimen and place in a CYTO-RICH collection vial and send it labeled with the patient's name and medical record number to the Cytopathology Laboratory with a completed requisition. Collapsible bags, syringes, or pleura-vac container are not acceptable. Normal volume: 1-50 ml.

Bronchoalveolar Lavaqe: For diagnosis of opportunistic respiratory infections The BAL is the preferred type of specimen; theusensitivity is greater than that for washings and brushings. (Sputum has a much lower yield, and is virtually useless for the detection of Pneumocystis carinii.)

Collect the bronchoalveolar lavage specimen in a clean, leak-proof, rigid container and deliver directly to the Cytopathology Laboratory with a completed requisition IMMEDIATELY after collection. Please be certain to indicate on the requisition if an opportunistic infection is suspected so that appropriate staining will be done.

Buccal Smears for BARR BODIES: For the detection and count of Barr bodies: buccal smears should be taken from above the midline of the inside of the cheek. Use a tongue depressor to scrape buccal mucosa forward several times, collecting "milky" cellular material. Quickly smear the material evenly on slide and immediately fix the slide in 95% alcohol. Repeat for 3 or 4 slides and send to Cytology laboratory with requisition. Be certain to state on requisition if specimen is for the purpose of Barr body evaluation.

Cerebrospinal Fluid: Collect the specimen in a clean, leak-proof, rigid container and send to the Fluids/UA Laboratory for distribution. Rush specimens may be brought directly to the Cytopathology Laboratory, but the Fluids lab should be informed that Cytopathology has been given their sample when the rest of the specimen is delivered there. Because cellular degeneration of CSF specimens begins within 20 minutes of collection, it is imperative that these specimens be delivered to the Cytopathology Laboratory as soon as possible. Please include pertinent clinical history (e.g. known or suspected malignancy, therapy history, etc.) on the cytopathology requisition in addition to the Fluids lab form for CSF's. If immunophenotyping is needed, a separate specimen should be sent to the flow cytometry lab.

Cervical/Vaqinal Smears

Sure Path Pap Test:

1. Obtain Sure Path collection vial from Histology and label with the patient’s name. 2. Vigorously swish the plastic spatula, brush or broom in the fluid. 3. Cut brush or broom off into the vial. 4. Make sure lid is tight (if fluid leaks out the cells may be lost). 5. Send fluid-filled vial with requisition to the laboratory.

Traditional (Conventional) Papanicolaou Smear Technique:

1. Use the following for one-slide – 2 spatula, 1 cytobrush, and cytology spray fixative. 2. Label the frosted end of the glass slide in pencil with the patient’s name. 3. Scrape the upper lateral vaginal wall lightly with a wooden spatula and have a nurse or assistant hold this spatula. 4. With a second spatula, lightly scrape the entire ectocervix, especially the borders of erosions and have a nurse or assistant hold this spatula. 5. Insert a cytobrush into the endocervix through the device, immediately roll the endocervical specimen contained on the cytobrush on the section of the slide farthest from the frosted end; quickly take the ectocervical specimen (second spatula) and spread the cervical material in the middle section of the slide; and finally take the vaginal specimen (first spatula) and smear it on the section of the slide closest to the frosted end. 6. Spray immediately with cytology spray fixative (caution – flammable)

The patient should be instructed not to douche for 24-48 hours prior to the examination. Please collect cytology specimens before manual examination, introduction of chemicals, biopsy, or before another procedure is performed. Lubricant should not be used on the speculum as it interferes with the staining reactions.

On the cytopathology requisition form, fill in the patient's name, age, and address, as well as the physician's name and location. Be sure date of specimen is indicated. Adequate clinical information is essential for proper interpretation of the specimen. Be sure to include complete relevant history, including date of last menstrual period, history of cancer or dysplasia, therapy history, gyne surgery, laser treatment, DES exposure, etc.

Sampling:

Cervical smears: Gently remove excessive secretion or blood at cervical os with cotton swab. Scrape cervix circumferentially at squamocolumnar junction. Spread material evenly onto one half of labeled slide. Then gently insert brush into cervical os. Rotate it. Retrieve brush and spread material evenly onto the other half of the slide. (It does not matter if the two samples mix together.) Fix with spray or alcohol IMMEDIATELY before any drying occurs. Vaginal and vulval smears: Fix and label.

Processing:

Allow fixative to dry before enclosing in the cardboard mailer. Label cardboard mailer with patient's name. Close mailer and secure with tape or rubber bands. Send with completed cytopathology requisition to specimen receiving/Cytopathology Laboratory.

Cause for Rejection:

Improper labeling of the slide or the requisition, fixation in formalin, drying of smear before fixation, incomplete requisition, failure to provide AGE, LMP, and history on requisition will result in rejection.

Colonic Brushings: Same collection procedures as for bronchial brushings.

Esophaqeal Brushinqs: Same collection procedure as for bronchial brushings.

Esophaqeal Washinqs: Same collection procedure as for bronchial brushings.

TZANCK Prep:

Note: Miscellaneous lesions for examination for herpetic viral changes. COLLECTION: Use frosted end of slides and label with the patient’s name. After the lesion has been scraped, take the applicator (cotton swab, wooden spatula, etc.) and rotate lightly on the top of the slide so that the material is evenly spread. IMMEDIATELY SPRAY FIX THE SLIDE WITH CYTOLOTY FIXATIVE. Allow the slide to air dry for several minutes. Place the slide(s) in a cardboard slide carrier, label carrier, and attach a completed cytology requisition labeled as Tzack Prep. Please indicate the EXACT SITE of the lesion and include any pertinent clinical history. Deliver the slides to the lab immediately.

REPORT: Interpretation by Pathologist. Minimum turnaround time is 48 hours.

Fine Needle Aspirations

A pathologist performs fine needle aspirations with rapid on-site diagnosis of superficial lesions on in-patients and out patients. Phone 423-431-6389 for scheduling.

FNA's in the clinic are available immediately upon clinician request or can be scheduled.

The Fine Needle Aspiration Service is available five days a week (Monday through Friday) between 8:00 AM and 5:00 PM.

To schedule the FNA, call 423-431-6389

The deep lesions requiring a radiographic guidance are performed by a radiologist in the Radiology Department. To assure the adequacy of the aspirate, the cytopathologist is always present at the time of procurement of the material. Contact the Radiology Department for scheduling.

Aspirations performed by clinicians

1. Determine the gross characteristics of the mass to be aspirated including location relative to other structures, estimated depth, consistency, and any evidence of pulsation or bruit. 2. Assemble the syringe and syringe pistol with attached needle (25 -23gauge). 3. Layout several glass slides, fixative and rinsing solution (Cytolyt or RPMI). 4. Label slides (using pencil), fixative and rinsing solution containers with patient’s name. 5. Check the requisition form if properly filled out - patient's name and Medical record number, attending full name, clinical information, site of aspiration. 6. Clean the skin over the puncture site with an alcohol skin preparation pad. 7. Grasp the mass firmly with the free hand and insert the needle in one swift motion. 8. Apply full vacuum pressure to the syringe with the pistol finger. 9. Move the needle back and forth within the mass at slightly different angles while full pressure is maintained. 10. Observe the junction of the needle and syringe for the appearance of any sample or continue to make back-and-forth passes within the mass about a dozen times. 11. Conclude the aspiration if the hub of a needle is filled with a material or after about dozen passes by releasing the trigger of the syringe pistol. DO NOT HOLD PRESSURE WHILE WITHDRAWING! If vacuum pressure is applied while withdrawing the needle, the sample will be pulled into the syringe where it will dry and be difficult to retrieve. 12. Withdraw the needle from the mass and place pressure on the puncture site with a sterile gauze pad. 13. Detach the needle from the syringe, fill the syringe with air and reattach the needle. 14. Express a small drop of aspirated material on one slide and gently spread the material by using a second slide. 15. Immediately fix one slide by dropping it into 95% alcohol. The second slide leave for air drying. 16. The remaining material, express into a rinsing solution. Rinse the syringe well. 17. Stain air-dried slides with Diff Quik. 18. Perform additional pass/passes until adequate material is obtained. 19. Bring all material (slides and rinsing solution) to the cytology laboratory for processing. 20. If the aspirated lesion is cystic, aspirate as much fluid as possible and place in a clean, leak proof rigid, labeled container. 21. Respirate any residual mass or a wall of a cyst and prepare smears as directed above.

Gastric Brushings:

Following same collection procedure as bronchial brushings.

Gastric Washinqs: Collect the specimen in a clean, leak-proof, rigid container. Patient must be fasting at least 12 hours before procedure. Evaluation for a NEOPLASM: Collect resting gastric contents and discard. Then instill 300 ml to 500 ml of physiologic saline solution, through the gastric tube. Have patient then sit, lie on back, lie on stomach, lie on right side, and lie on left side. Aspirate as much of injected saline as possible and place in a clean leakproof container. Deliver IMMEDIATELY labeled with the patient's name and medical record number and with completed requisition to specimen receiving or the Cytopathology Laboratory.

Scrape/Smear (Eve. Oral. Skin): Label the slide with the patient's name. Scrape the lesion with a tongue blade or metal spatula. If the skin lesion is a vesicle, break vesicle and scrape around edges of ulcer, not the center. Spread scrapings rapidly and evenly on glass slide. Fix slide IMMEDIATELY with spray fixative or drop in a jar of 95% ethyl alcohol. Deliver in slide holder or jar with completed requisition to specimen receiving or the Cytopathology laboratory.

Serous Fluids (Pericardial. Peritoneal. Pleural): Collect the specimen in a clean, leakproof, rigid container (no vacuum bottles, drainage bags, syringes with needles attached, etc.). Cover tightly, label with patient's name and medical record number, and send fresh specimen to the cytology laboratory. In case of copious amounts of fluid, a 100cc well-mixed sample will suffice for cytological evaluation. NOTE: Please do not use the "CSF and Other Body Fluids" form in place of the Cytology form for fluid specimens. A completed cytopathology requisition is required for all specimens submitted to the cytology laboratory, even if a fluid lab form is also filled out for other tests. The importance of filling in complete history (known or suspected primary cancer, chemotherapy, radiation, clinical symptoms, surgery, etc.) cannot be overemphasized, and is essential for complete evaluation of a cytologic specimen. Small Intestinal Brushings:

Same collection procedure as for bronchial brushings

Sputum: Collect an early morning deep cough specimen (not just saliva) in a clean, leakproof, rigid container labeled with the patient's name and medical record number. The mouth must be rinsed out thoroughly prior to sputum collection to avoid contamination with food. Specimens must be delivered fresh to the cytology laboratory or refrigerated until delivery. NOTE: In order to assure the maximum diagnostic benefits afforded by sputum cytology, it is essential to submit early morning deep cough specimens. These should be collected for at least 3 consecutive days. The patient should be informed as to the difference between sputum and saliva and instructed to rinse out his mouth first. Outpatients may be provided with containers of fixative in which sputum is to be collected, so that they may collect the samples on 3 days and bring them in all at once without concern for degeneration. Inpatient sputum samples are to be sent fresh to the laboratory, without fixative.

Instructions to Patients for Sputum Cytology:

This is a special test which requires a fresh morning specimen of sputum, collected on three separate days. A separate container of preservative is to be used each day. THE FLUID IN THE CONTAINER IS A TOXIC SOLUTION AND IS NOT FOR HUMAN CONSUMPTION.

It is essential that you follow the simple instructions given below:

 SPUTUM is the material from deep down in the chest and can ONLY be obtained by deep coughing.  SALIVA is the watery fluid in the mouth and is not desired in this test.  POSTNASAL DRAINAGE is the thick material, which may drain down form the nose and collect in the back of the throat, especially during the night. If this is present, it should be cleared from the throat and DISCARDED before collecting the sputum specimen.

INSTRUCTIONS:

1. On the morning the specimen is to be collected, before breakfast, clear your throat and discard this material. IT IS ESPECIALLY IMPORTANT THAT YOU RINSE OUT YOUR MOUTH WITH WATER FIRST, IF YOU HAVE TAKEN ANYTHING BY MOUTH - such as food, milk, coffee, mouthwash, toothpaste, etc. Do not contaminate specimen with tobacco ashes, Kleenex, etc. 2. Breathe deeply 8 to 10 times, cough deeply and expectorate into the container provide. Firmly tighten the lid and shake container vigorously for a few seconds. Collect the sputum obtained this way during the first half-hour. A small amount of sputum will be sufficient for study, IF IT HAS BEEN RAISED FROM DEEP IN THE CHEST. Do not submit a specimen unless you believe it is sputum from deep in the chest.

Instructions to Outpatient Clinic for Sputum Collection:

1. Give the patient an instruction sheet and three containers filled with 50 ml. of the fixative provided by cytology (Saccomanno fixative). 2. The instruction sheet explains to the patient the method of collection. 3. Tell the patient that he/she is to collect three separate sputum specimens at home on three consecutive days and return them to the clinic all together after the third one has been collected. 4. Return the containers to the lab with completed pink cytology requisition forms. Be sure each container and requisition has the date of specimen collection.

Tracheal Brushings: Same collection procedure as for bronchial brushings.

Tracheal Washings: Same collection procedure as for bronchial brushings.

Urine – Bladder – Urethral - Renal Pelvic: Send a fresh 5-100ml sample in a clean, leak proof container labeled with the patient's name and medical record number to the cytology laboratory as soon as possible. Due to the deleterious effects of an acid environment on cell preservation, 24-hour samples are not acceptable for cytologic evaluation. Please refer to the section on hours of the cytology laboratory to ensure that samples are sent during work hours when they can be processed fresh. Specimens unable to be delivered during laboratory hours should be placed in a refrigerator (but not allowed to freeze) and delivered as soon as possible the following workday. No anticoagulant or fixative is to be added. Label the container as applicable to specimen source. Indicate on the cytology form whether the urine is voided, barbotage, catheterized, or from cystoscopy.

Specimens for metachromatic leukodystrophy must be delivered immediately after collection. CytoLyt® or Preservcyt® may be added for transportation purposes. The cells may degenerate of CytoLyt® is used as a permanent fixative. Ideally the cells should be fixed immediately in Presevcyt® solution at collection site.

Reasons for Rejection/Unsatisfactory Results: Improper labeling of either specimen or requisition, addition of anticoagulants or fixatives to the specimen, incomplete requisition (including failure to provide clinical history), allowing specimen to freeze, or improper fixation in the process of making smears from unprepared specimens.