CERVICAL, VAGINAL, AND ENDOCERVICAL SPECIMEN

Principle

To establish a uniform method of preparation that will be an aid in the diagnosis of genital abnormalities, infections and an aid in the evaluation of hormonal functions for all gynecological specimens (Thin-Prep and conventional smears).

1. Criteria for Acceptance of specimen
   1. All vials and slides should be labeled with name of patient and a unique ID (MRN, Social Security or birthdate).
   2. A completely filled out requisition should accompany the specimen to include Name, LMP, and Clinical History.
2. Patient Preparation
   1. The patient should be instructed to avoid the use of douches, vaginal medications or spermicides 48 hours prior to testing. A 24-hour abstinence from sexual intercourse should be maintained.
   2. Please remember that the best time to perform a “Pap” smear is two weeks after the first day of LMP (day 14 of the cycle). Pap smears collected during menses are useless for interpretation
   3. Avoid submitting pap smears in the immediate post-partum period (less than six weeks from delivery). If there are some purulent exudates, treat the infection prior to obtaining a pap smear.
3. Preparing the patient
   1. Before inserting the speculum, remember to lubricate it using warm water. Avoid usage of lubricant jelly if at all possible, since this material often obscures the cells on slides, therefore hampering accurate interpretation.
   2. Sampling of cervix is paramount for specimen adequacy. Cells from this area (in particular squamo- columnar junction) are often the first to show pathologic changes
   3. Be aware that excessive blood, mucus or inflammatory material should be removed (from cervix) with dry gauze to enhance the quality of the smear.

CONVENTIONAL SMEARS

Materials required

Pap pak kit includes: scraper, cytobrush, fixative and glass slide with one end frosted for identification of patient (write with pencil), Cardboard folders, slides, cyto brushes, scrapers and fixative

Collection technique

1. Tear open fixative pouch or prepare spray bottle
2. Label slide clearly with the patient’s name on frosted end. Use a #2 pencil. Do not use ink or felt tip marker pens as they are soluble in staining procedure
3. Prepare smears as illustrated on Pap-pak
4. Take cervical smear by rotating the cervical scraper around ectocervix with special emphasis on the squamo-columnar junction. Spread material evenly on Section “C”.
5. Take endocervical smear by gently inserting cytology brush into endocervical canal past squamo-columnar junction. Rotate cytology brush one full turn (360°). Gently remove cytology brush without touching vaginal surfaces. Spread material evenly by rotating cytology brush back and forth over Section “E”.
6. Take vaginal smear from posterior fornix with spatula end of cervical scraper. If an accurate hormonal evaluation is desired, obtain specimen by lightly scraping mid-lateral vaginal wall. Spread material evenly on “V” section.
7. Immediately fix preparation by flooding entire VCE slide with fixative. Proper fixation is necessary and important to assure clear nuclear detail. Let air dry 15 minutes.
8. Label the slide holder and wrap the cytology slip around it. Secure with rubber band.
9. If a maturation index is required, a separate slide must be submitted from the lateral vaginal wall. Maturation indices cannot be performed on slides with endocervical components.
10. When using a cytobrush, avoid vigorous friction, which results in bleeding and distortion of cells. Sampling of the endocervical canal should be accompanied by a sample of the Ectocervix.

WARNING
The cytology brush should not be used on pregnant patients
The cytology brush should not be used to sample endometrium

THIN-PREP SMEARS

Materials required

Thin-Prep Vial
Spatula
Brush

Collection Technique

1. Label the vial with patient name or ID
2. Complete cytology requisition to include; patient name, LMP, date of collection and revelent history.
3. Use plastic spatula to obtain an adequate sampling from the ectocervix. Rinse the spatula as quickly as possible into the PreservCyt solution vial by swirling the spatula vigorously in the vial 10 times. Discard the spatula.
4. USE THE BRUSH to obtain an adequate sampling from the endocervix. Insert the brush into the cervix until only the bottom-most fibers are exposed. Slowly rotate ¼ or ½ turn in one direction. DO NOT OVER ROTATE. Rinse brush quickly as possible in 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. To obtain an adequate sampling from the cervix using the broom-like device. Insert the central bristles of the broom into the endocervical canal deep enough so all the shorter bristles can fully contact the ectocervix. Push gently and rotate the broom in a clockwise direction five times. Rinse broom as quickly as possible 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 vigorously to further release material. Discard the collection device.
6. Tighten the cap so that the torque line on the cap passes the torque line on the vial.
7. Place vial and matching Cytology requisition in a specimen bag for transport to the Laboratory.

NOTE: ANAL Smears are also collected in Gyn Thin Prep vials but are processed as Non-Gyn Specimens and given a Non-gyn number.

NIPPLE SECRETIONS

Principle
The chief value of the cytologic examination of the nipple secretion lies in the possible early diagnosis of asymptomatic cancer of the ducts of the breast and benign disorders such as duct stains or papilloma. This procedure should be confined to those patients who have no palpable masses in the breast or other evidence of breast cancer.

Materials
Clean glass slides
Paper clips
Glass container with a 95% alcohol solution or cyto spray-fixative
Cytology requisition
Cardboard slide folder (if spray fixative is used)

Procedure

1. Label several clean glass slides with patient’s name, date and type of specimen. Paper clip every other slide (if 95% alcohol solution is used).
2. Open a bottle with fixative (95% ethyl alcohol) and have the patient hold the open bottle below the breast
3. GENTLY express only the nipple and subareolar area using the thumb and forefinger. If no secretions appear at the nipple with the gently compression, do not manipulate further.
4. If secretion occurs, allow only a drop the size of a pea to accumulate upon the apex of the nipple. If a drop is obtained, it could be smeared on the slide
5. Immobilize the breast with one hand
6. With the other hand place slide upon nipple, momentarily pause to allow the material to spread a bit laterally, then draw the slide quickly across the nipple
7. Immediately drop slide into bottle of fixative or saturate with cytology spray-fixative
8. Repeat complete procedure and make as many smears as the secretion obtainable from the nipple allows
9. Submit to laboratory

FINE NEEDLE ASPIRATION AND NON-GYN SPECIMENS

Purpose
A variation in normal cellular morphology is the precursor to most malignancies and a feature of malignancy. By aspirating and sampling various tissues and processing smears or cytospin slides according to a Papanicolaou procedure, it is possible to identify these changes and give an “early” diagnosis for probable malignancies or diagnosis of overt malignancy.

Specimen
Non-gyn specimens

Procedure
Specimens are received in several forms

1. One slide or multiple slides smeared with samplings of the desired area
   
   1. The patient’s name must be written of the frosted end of the slide
   2. On the slide(s), prepare in a thin, uniform cellular spread
   3. The smear(s) must be sprayed with Cytology Fixative IMMEDIATELY, before the slightest trace of air drying can occur
   4. A completed requisition form must accompany the specimen
2. Aspirated material from cysts, solid lesions, etc.
   
   1. Aspirated fluids are submitted in cytology preservative/transport fluid and submitted to the cytology department with a completed cytology requisition
   2. The fluid is centrifuged at 2000 rpm for 10 minutes, pour off the supernant
   3. If the cell button is visualized, prepare 4 direct smears using the standard smear technique
   4. If the sediment is scanty, prepare a cytospin using the cytocentrifuge technique
   5. Any remaining specimen is submitted for a cell block, especially any clotted material

Procedure notes

A specimen is considered unacceptable if:

1. Slide(s) badly broken
2. Name on slide doesn’t match name on the requisition
3. Slide is not labeled with the patient’s name
4. Tube or container has leaked its content

SPUTUM COLLECTION

Principle
When a pulmonary lesion is suspected, a complete sputum series should be examined. The COMPLETE SPUTUM SERIES is an EARLY MORNING SPECIMEN EACH DAY FOR FIVE DAYS. The complete sputum series increases the detection of primary bronchogenic carcinoma from 45% (one specimen) to 95% (five specimens). DO NOT SUBMIT 24 HOUR COLLECTIONS.

Specimen
Sputum- Deep cough or post bronchoscopy

Materials
Wide mouth plastic container with screw top

Procedure

1. Sputum Technique
   1. Give the patient one of the above plastic containers, adequately labeled, the night before and instruct the patient not to use it until morning
   2. Instruct patient to cough DEEPLY, upon awakening, first thing in the morning, (before meals or before brushing teeth) and expectorate all sputum into the container. Encourage the patient to expectorate SPUTUM and not SALIVA.
   3. The patient should continue the deep coughing intermittently for about one-half hour. In this time an adequate specimen generally is collected.
   4. Repeat the procedure each day for a minimum of three days and preferably for five days.
2. Post-Brochoscopy Technique
   1. Give the patient one of the plastic containers mentioned above, BEFORE the bronchoscope is withdrawn
   2. Have the patient cough deeply and expectorate all sputum into the container for 1-2 hours
   3. Continue the sputum series using Sputum Technique
3. For patients with BRONCHIAL BLOCKAGE, heavy INFLAMMATION, or previous INCONCLUSIVE reports: When there is a suspicious chest x-ray and no malignancy is evident on cytology examination but there are changes consistent with an obstructive process we would suggest a repeat series after adequate therapy (antibiotic, expectorant and/or bronchodilator)
   1. Administer therapy for 5 days
   2. On the third day of this combined therapy, commence the sputum series as in sputum technique

URINE COLLECTION

Principle
Multiple voided urine specimens are invaluable in assessing the status of the lower urinary tract. Catherized urine is acceptable. For cytological evaluation of the bladder, three morning samples of urine, each about 50-100 mL, obtained consecutive days are recommended.

Specimen
Urinary tract

Materials
Clean plastic urine container
Cytology requisition

Patient Preparation

1. The first voided urine of the day is the poorest urine for cytology examination, due to degeneration of urothelial cells. It should be discarded.
2. Drink plenty of water (or tea, coffee, fruit juices, etc.) every 15 minutes for at least 2 hours
3. Void at no will into the container provided. Female patients should use a tampon or gauze to prevent vaginal contamination
4. Continue drinking fluids and voiding at will until 200 mL or urine (about 1 cup) or more has been collected
5. This entire procedure should be repeated for at least 3 days (not necessarily consecutive days)

Procedure

1. Label several clean glass slides with patient’s name, date and type of specimen.
2. Brush the surface of suspicious areas completely
3. Withdraw the brush, quickly roll it on a labeled glass slide, which is held over an open bottle of 95% alcohol fixative and immediately drop the slide into the fixative. Immediate fixation is essential to AVOID DRYING which occurs rapidly after material is spread onto the slide.
4. Submit to laboratory

CEREBROSPINAL FLUID COLLECTION

Purpose
Cerebrospinal fluid specimens are utilized to detect primary or metastatic lesions to the brain as well as detecting possible infection or autoimmune diseases that may be ensuing on the patient. The volume of the samples has considerable bearing on diagnostic accuracy, the larger the sample, the better the results. If several samples are obtained, the second or third should be used for cytology.

Equipment
Clean sealed glass container
Cytology Requisition

Procedure

1. The patient’s physician should collect the fine needle aspiration specimen utilizing their own training and procedures.
2. The aspirated cerebrospinal fluid should be placed in a clean test tube or container.
3. Fill out the cytology requisition completely.
4. Include all the patients’ information necessary for the laboratory to make an accurate identification. This includes:
   
   1. First and last name
   2. Date of birth
   3. Source of specimen
   4. Pre- op diagnosis
   5. History, if applicable
5. Send specimen, with its completed requisition form, to the laboratory.

SEROUS FLUID COLLECTION

Purpose
The preparation of serous fluids for microscopic examination is utilized to detect the presence or absence of abnormalities in the serous cavities such as, rheumatoid arthritis, lupus, endometriosis, and malignancy (whether primary or metastatic). Therefore, aiding in diagnosis, proper treatment, and care for the patient.

Specimen Type
Pleural Fluid
Peritoneal Fluid
Pericardial Fluid

Equipment
Clean leak-proof collecting container.

Procedure

1. The patient’s physician should collect the fine needle aspiration specimen utilizing their own training and procedures.
2. Label the leak-proof container and fill out cytology requisition.
3. Collect at minimum of 2 ml of body fluid.
4. Do not add any fixatives or anticoagulants to the patient’s specimen.
5. Fill out the cytology requisition completely.
6. Include all the patients’ information necessary for the laboratory to make an accurate identification. This includes:
   
   1. First and last name
   2. Date of birth
   3. Source of specimen
   4. Pre- op diagnosis
   5. History, if applicable
7. Send specimen, with its completed requisition form, to the laboratory.
8. Fluid is to be refrigerated at the laboratory until preparation is done.

ANAL SMEARS

Anal smears are collected in Thin-Prep, Sure-Path containers and can also be collected as a pulled smear.
Specimens should be accessioned and processed as a non-gyn specimen

PLEASE NOTE: Any questions about collection of any specimen, please contact the laboratory during the day and ask to talk to someone in the Cytology Department.