Procedures for Specimen Preparation and Obtaining Pathology Services

Autopsy

INPATIENTS: PMA pathologists will perform autopsies on MMC inpatients when the attending physician requests one, except for cases of Jacob-Creutzfeld disease or certain other high risk diseases. If a staff physician is interested in having an autopsy performed on a patient, she/he must get permission of the next of kin (spouse>parent/child>sibling>other relative assuming responsibility for the body). The next of kin must sign the MMC Autopsy Permit Form, which can be obtained from the nursing staff. The attending doctor should contact the pathologist on call to communicate the desire for an autopsy and to pass on any relevant clinical information or special requests.

In cases where the family requests that an autopsy be performed, the MMC nursing staff or next of kin should contact the pathologist on call to discuss the reasons for the autopsy. The next of kin must assume financial responsibility for the autopsy by signing the Autopsy Financial Responsibility Form as well as the MMC Autopsy Permit Form. The pathologist on call will decide whether or not an autopsy will be performed on cases requested by a family. All reasonable attempts will be made to honor appropriate family requests.

OUTPATIENTS: All requests for autopsies on patients who are not MMC inpatients will be handled in the same manner as for family requested autopsies on inpatients.

All autopsies are performed in the morgue at MMC usually between the hours of 8 AM and 4 PM seven days a week. Attending physicians or others involved in the care of the deceased may attend the autopsy or be notified when the autopsy is completed to review the gross findings. A Preliminary Autopsy Report will be issued or called to the attending physician within 24 hours of the completion of the autopsy. A Final Autopsy Report will be sent to the deceased's physician within 30 days. In addition, a copy of the Final Autopsy Report will be sent to the MMC Health Information Department for all inpatient autopsies. For outpatient autopsies, a copy of the Final Autopsy Report will be sent to the family along with a letter from the pathologist explaining the major findings.

Brushings (bronchial, etc.)

Smears should be made as soon as the specimen is obtained on the brush. In a circular motion make a smear about the size of a quarter in the center of a slide with the brush, without excessive pressure or trauma to the smear. Immediately drop the slide in a bottle of 95% alcohol fixative before any drying can occur. Placing the brush in fixative and/or making multiple slides are not recommended.

Cervical Cone (orientation)

Cervical cone excision specimens should be opened parallel to the endocervical canal and carefully pinned flat on a tongue depressor. The location where the cone was opened should be indicated on the requisition form to aid in orientation of the specimen (e.g. "Opened @ 3 o'clock"). The endocervical end of the specimen should be indicated using dye on the tongue depressor, a suture or other means. These precautions are necessary for pathologists to be able to report on the correct location and margins of lesions.

Consultation, pathologist

A pathologist is available for consultation on clinical pathology matters 24 hours a day, 7 days a week in an emergency. Some tests, such as Protein Electrophoresis, Blood Bank Antibodies and a few others, are always interpreted by a pathologist to aid the attending physician in making a diagnosis or treating the patient. Consultation on test selection, test interpretation, peripheral smear results or any other laboratory matter may be requested by a physician. For interpretation of test results, help with test selection or other complex laboratory questions, a physician may consult a PMA pathologist in the same manner as any other specialist. For review of a peripheral blood smear, a Blood Smear Review may be ordered as a specific test in the MMC computer or on a surgical pathology requisition. Whenever appropriate, the consultant pathologist will issue a report summarizing the consultation.

Crystals (joint fluid)

Joint fluid should be collected in either liquid EDTA (purple top tube) or heparin (green top). Submit the fluid to the laboratory with tissue requisition or computer orders for either Crystals or the complete Joint Fluid Analysis. The specimen will be examined by polarized light microscopy with compensation, if indicated.

DNA Ploidy analysis, bladder washing:

Bladder washings may be analyzed for the presence of aneuploidy and hyperdiploidy by flow cytometry. The washing should be obtained by irrigation with several vigorous pulses of sterile saline and placed into a labeled, leakproof container and refrigerated. A minimum of 15-30ml (preferably 30ml) of bladder washing should be obtained and must be received by the reference laboratory within 24 hours.

DNA Ploidy analysis, breast tissue:

Breast tumors may be analyzed for aneuploidy and % S phase by flow cytometry. 1-2 grams (preferably 2 grams) of fresh tissue should be obtained and refrigerated soon after surgical resection. A room temperature, formalin-fixed paraffin block may also be sent for analysis.

DNA Ploidy analysis, hematologic:

Blood, bone marrow, and lymphoid tissues may be submitted for DNA ploidy analysis. 1 to 2ml (preferably 2ml) of blood and bone marrow aspirates should be submitted to the clinical laboratory at room temperature in a sodium heparin tube. Lymphoid tissue should be submitted fresh to the pathologist on call so the tissue may be minced and placed in a special transport media. Do not refrigerate. The specimen must be received by the reference laboratory within 24 hours.

Extremity, amputation:

Amputated extremities, usually above or below the knee amputations of the leg, are to be placed in a red plastic bag, sealed, and labeled with the patient’s name and other identifying information. No fixative is necessary. A surgical requisition form must be filled out with relevant diagnostic and clinical information and taken to the laboratory. The labeled extremity is to be taken to the morgue and placed in the bottom cooler.

Fine Needle Aspiration Biopsy

Detailed description of the technique of fine needle aspiration biopsy is beyond the scope of this manual. However, the following points are important to remember.

Use aseptic technique.
Apply the minimal amount of suction required to visualize material in the hub of the needle. This suction is applied after the needle is in place.
Vary the angle of the needle when moving through the lesion.
Release the suction before withdrawing the needle.
Rapidly prepare and fix slides or express into fixative (make sure to flush the needle or hub) to prevent air drying artifact and clotting.
At least three separate passes from different angles are recommended.
Place material from at least one pass in 10% formalin for cell block preparation.
Do not submit syringes with the needle attached.
Specific situations may require slight alteration of technique or specimen handling. For example:

1. Cysts: When cysts are encountered an attempt should be made to evacuate and collapse it. Slowly increase the suction and move the needle in a wide cone while pushing on the mass. Also, when a cyst is aspirated withdraw the needle with some vacuum in the syringe. The cyst fluid should be placed in an equal part of “cytology fixative for fluids” (cytolyte).

2. Thyroid Aspiration: Use a 25-gauge needle as larger needles introduce too much blood. Make one air dried smear (second smear) and fix at least one to two slides (by immersion into 95% alcohol, “cytology fixative for smears”). Place additional material in 10% formalin (for cell block preparation).

3. Breast Aspirations: Make smears and fix immediately in 95% alcohol, (“cytology fixative for smears”). Place additional material in 10% formalin for cell block. Do not use “cytology fixative for fluid” (cytolyte) unless a cyst is encountered.

Evaluation of fine needle aspiration specimens are available on the same schedule as other nonGYN cytology and results are generally available 24 hours after receipt of the specimen. Specimens received after 2:00 p.m. will be processed the following day.

Fine Needle Aspiration, Immediate Evaluation (With Radiology)

The purpose of this procedure is to determined the adequacy of the sample and to triage the aspirated material to allow for best use of the sample.

Notify the pathologist scheduled for cytology of the approximate time of the procedure. Make arrangements at that time regarding second notification (generally 10-15 minutes before the specimen is aspirated).

When either a lymphoma or mesothelioma is suspected, notify the pathologist of that fact so that special preservatives can be thawed or prepared prior to sample retrieval.

Immediate evaluation of FNA’s are available between the hours of 8:00 a.m. and 5:00 p.m. Rarely, if there is a conflict with a frozen section, the frozen section takes precedence.

Flow cytometry for leukocyte markers:
Flow cytometry may be used for the detection of lymphoma or leukemia cells in blood, bone marrow, and/or tissue. For analysis from peripheral blood, 10 ml of heparinized blood and 5 ml of EDTA blood are required. At least 1 ml of bone marrow aspirate in heparin is needed. Lymph node tissue should be submitted fresh to the pathologist on call so the tissue may be minced and submitted in a special transport media. Do not refrigerate the specimen. If there is clinical suspicion of a specific type of lymphoma or leukemia, indicate on the requisition or order. If there is a strong clinical suspicion of acute leukemia, please notify the pathologist on call and/or referral testing so the specimen may be expedited for a faster turnaround time.

Fluid, Aspiration (for cytology)

Large volumes of fluid such as pleural or peritoneal fluid (more than 25-50 ml) should be brought to pathology as soon as possible without adding fixative. If immediate transportation is not possible, refrigerate the specimen until it can be transported to pathology.

Small volumes of fluid such as a breast cyst aspirate (less than 50 ml) should be mixed with an equal volume of Cytology Fixative. This may be done by aspirating an equal volume of fixative into the syringe with the specimen or by expelling the aspirate into an equal volume of fixative in another container.

Preparation of slides is not required.

If an aspiration of a suspected cyst produces only a minimal quantity of material in the hub of the needle, follow the procedure for preparation of slides in the section on Fine Needle Aspiration Biopsy.

FNA (See Fine Needle Aspiration)

Fresh Specimen
Tissue removed during surgery may be submitted fresh for immediate evaluation by the pathologist or for special studies. The tissue to be submitted should be removed and placed in a container with no fixative (small specimens should be placed on a telfa pad moistened with normal saline to prevent drying of the specimen). The container should be labeled with the patient's name. The specimen and properly completed Surgical Pathology Requisition should be immediately taken to the anatomic pathology area of the laboratory. All relevant clinical information should be listed on the Requisition to make sure that the appropriate special procedures (e.g. Flow cytometry, special fixatives, etc.) will be done.

Fresh Specimen with Culture

Tissue removed during surgery may be submitted for immediate examination by the pathologist following preparation of cultures by the laboratory. The tissue to be submitted should be removed using sterile technique, placed in a sterile container with no fixative and labeled with the patient's name. The appropriate cultures must be ordered in the MMC computer and a surgical pathology requisition must be properly filled out. The specimen and requisition should be immediately taken to the laboratory. After the microbiology laboratory has prepared the appropriate cultures, the remaining specimen will be delivered to the anatomic pathology laboratory for examination by the pathologist, where the appropriate special procedures (flow cytometry, special fixatives, etc.) will be done, as indicated, as well as routine tissue examination.

Frozen Sections:

Frozen sections are submitted from the operating room according to the procedure in the operating room manual. If frozen sections are submitted from other sources, advance notification of the pathologist on call is essential. For all frozen sections, page the pathologist on call as soon as the specimen is ready for transport to the laboratory, or as soon as the need for frozen section becomes apparent if the case is unscheduled. The tissue is placed in a container without fixative. A surgical pathology requisition form and consultation form must be filled out with relevant diagnostic and clinical information and taken to the laboratory, where the requisition will be time-stamped and the Surgery record of receipt will be signed by lab personnel.

Gene rearrangement:

Gene rearrangement by Southern blot analysis may be useful in the detection of clonality of certain lymphomas. Prior notification to referral testing in the MMC Clinical Laboratory is recommended. Blood and bone marrow aspirate specimens may be submitted in heparin and refrigerated. Tissue specimens should be sent fresh to the pathologist on call. The tissue will be placed in RPMI media (yellow cap frozen “Transport media” in referral testing), refrigerated, and sent for analysis. Specimens for gene rearrangement analysis should be obtained Monday through Thursday.

Genetic analysis of fetal tissue:

Fresh sterile tissue from stillborns, fetuses, products of conception, or malformed infants may be analyzed for the presence of chromosomal abnormalities. The attending physician obtains the tissue samples, places them in a sterile container with enough sterile saline to just cover the specimen, and submits them to the referral testing section of the laboratory. The specimen should be refrigerated, not frozen.

Hormone receptors:

Estrogen and progesterone receptors are typically performed on a breast biopsy or excision and reported with the diagnosis on the surgical report. Unless otherwise requested, these will automatically be performed on all invasive breast carcinomas. Previously diagnosed cases of breast carcinoma and tissue from metastatic tumor may have hormone receptors performed on recut sections of paraffin blocks upon request, Monday through Friday. The results will be appended to the original pathology report, which is then reissued to the physician.

Lymph Node (for Lymphoma)

Lymph nodes removed at surgery for suspected lymphoma should be handled in the same manner as a Fresh Specimen. In addition, any requested special tests (e.g. flow cytometry) should be indicated on the requisition. The pathologist will examine the specimen and set up any special fixatives or additional procedures that may be needed.

Muscle biopsy:

Muscle biopsies for muscular dystrophies and other primary muscle diseases require specialized enzyme studies which are performed at the University of Tennessee. These specimens are to be sent directly to the MMC clinical laboratory, reference laboratory section. Immediately upon excision the specimen is placed on a tongue blade with as little handling as possible, wrapped in sterile saline-soaked gauze, and placed in a clean, dry container. The container must be accompanied by all essential clinical information, including symptoms, diagnosis, duration, and the results of EMG and other testing.

Pap Smear Conventional

The optional time for collection of the pap smear is approximately two weeks (10-18 days) after the first day of the last menstrual period. The patient should be instructed not to use vaginal medications, spermicides or creams 48 hours prior to collection and not to douche for 24 hours prior to collection. The patient should also refrain from intercourse 24 hours prior to collection of the pap smear.

The cytology request form should be completed just prior to taking the sample. The form should contain the following information:

Patient’s name (any name change in the past five years should be noted).
Age, date of birth, and social security number.
Menstrual status (LMP, hysterectomy, pregnant, post partum, hormone therapy).
Previous abnormal result, treatment, or biopsy.
Patient’s risk status (e.g. “high risk”).
Relevant history (contraceptive used, abnormal bleeding, etc.).

The frosted, or colored end of a glass slide should be labeled in pencil immediately prior to collection. The slide should be labeled with the patient’s first and last name.

With the patient in the dorsolithotomy position, expose the complete face of the cervix using a vaginal speculum lubricated using warm water only. The transformation zone is the site of origin for most cervical abnormalities and should be the focus of collection efforts. If the patient has had a total abdominal hysterectomy, a vaginal sample with special attention to the vaginal cuff is taken. After the cervix is visualized remove any excess blood, mucus, or inflammatory material gently with a dry gauze square or cotton-tipped applicator. Do not forcibly remove any cellular material.

Pap smears may be collected using a number of devices and techniques. The two most important steps in all cases are (1) adequate sampling of the transformation zone (squamocolumnar junction), and (2) rapid fixation of the smeared cellular material.

Endocervical Brush:
The endocervical brush is inserted into the endocervical canal until only the bristles closest to the handle are visible. The brush is rotated ¼ to ½ of a turn. The brush is removed and held until the ectocervical sample is retrieved. When both samples have been collected and the ectocervical sample has been smeared (see below), roll the endocervical brush across the glass slide with gentle pressure. Fix immediately by immersing in 95% ethanol or using the spray fixative. Use of an endocervical brush tip may be contraindicated in pregnant patients; refer to manufacturer instructions and clinical judgement.

Wooden or Plastic Extended-TIP Spatula:
This sample is taken after the endocervical sample. The notched end of the spatula that corresponds to the contour of the cervix (long arm in cervical os) is rotated one time around the circumference of the external cervical os and adjacent portio vaginalis. The cellular material is spread thinly over the surface of the glass slide. Immediately fix the slide by immersing in 95% ethanol or using the provided spray fixative.

Plastic “Broom-Type” devices:
The “broom-type” devices sample both the endocervix and ectocervix simultaneously. The broom is inserted into the cervical canal until the lateral bristles bend fully against the ectocervix. The sampling device is rotated 360 degrees in the same direction five times while maintaining gentle pressure. The broom is removed and with a single paint stroke motion, the cellular sample is transferred down the long axis of the slide. The broom is turned over and this is repeated over the same area. The slide is fixed immediately by immersing in 95% alcohol or using the spray fixative.

Preparation for Transport to PMA
Slides fixed in 95% alcohol may be transported in the container or, if preferred, the slide may be fixed in the alcohol for 20-30 minutes, removed, allowed to dry, and placed in a slide container for transport. The specimen (slide container, or bottle with slide), should be placed in a bag with a completed requisition for transport. Pap smear results are generally completed within a week days after specimen is received.

Pneumocystis

Specimens which are suspected to contain Pneumocystis carinii organisms should be collected and submitted in the same fashion and with the same fixatives as other specimens. The requisition should clearly indicate that identification of Pneumocystis organisms is being requested. The special stain for these organisms can be performed Monday through Friday, if begun before noon. Special arrangements may be made to perform the special stain on Saturday mornings in emergencies, provided that PMA has prior notification that the stain will be needed.

Renal Calculus analysis

Renal calculi for stone analysis should be submitted to the laboratory in a sealed clean container without any fixative, labeled with the patient's name. An order for a Kidney Stone Analysis should be made via computer. The specimen will be sent to our reference lab and results should be returned within 2 weeks.

Requisition (completion requirements):

A completed surgical pathology requisition slip MUST accompany the tissue or cytology specimen with the patient’s name, social security number, billing information, age, sex, race, pertinent clinical history, and source of specimen.

Pap smears should also include the date of the patient’s LMP, any hormonal therapy, a history of previous abnormal pap smears, and any pertinent treatments such as cryotherapy, conization, radiation, or chemotherapy.

Rush requests:

Results may be faxed or called to the physician if requested. Please write “RUSH” on the requisition form and fill in either the fax or phone number for result reporting. Most results are available by morning or early afternoon of the day of microscopic evaluation and will be called to the attending physician.

Sentinel lymph node
The sentinel lymph node is excised by the surgeon and should be placed in a container without fixative or other fluids, and labeled with the patient’s name and other essential identifying information. A surgical requisition form is filled out with identifying information, clinical history and diagnosis. The specimen and the requisition are then taken to the tissue processing area of the laboratory. The lymph node will be processed and examined according to the pathology special procedure for sentinel lymph nodes.

Skin scraping
Skin scrapings are performed to diagnose superficial fungal infection or herpesvirus infection. The scraping is usually performed by the physician with a scalpel blade, and the resulting sample is spread thinly on a glass slide and immediately placed in 95% alcohol. This bottle, which may be obtained from pathology, is then labeled with the patient’s name and other essential identifying information. It is then submitted to pathology with a cytology requisition form labeled with the patient’s name, clinical information and diagnosis.

Sputum For Cytology

Early a.m. sputum specimens yield the greatest number of diagnostic cells. Sputum for cytology should be collected separately from that for other analysis (microbiology, etc.).

The patient should be instructed to clear the throat of post nasal secretions and to gargle and rinse the mouth with water to remove food residue. The patient is then encouraged to cough deeply. Expectorated material is collected in a wide-mouth, appropriately labeled container, fresh or with fixative (see below).

Specimens collected at times when they cannot be processed by the lab within four hours or on nights or week-ends should have an equal volume of “cytology fixative for fluid (thin layer)” added. This should be noted on the requisition form.

An adequate cytologic evaluation consists of a series of 3-5 consecutive daily sputum specimens. Alternatively, a 3-day pooled sputum collection (kept in fixative) may be done. Patients that cannot spontaneously cough may have induced sputum.

Cytologic results are generally available within 12-24 hours after receipt of the specimen.

Thin-layer cytology, genital (e.g. Thinprep):

Broom-like device protocol:

1. Obtain an adequate sampling by inserting the central bristles of the broom into the endocervical canal deep enough to allow the shorter bristles to fully contact the ectocervix. Push gently and rotate the broom in a clockwise direction five times.

2. Rinse the broom as quickly as possible into the Preservative 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.

3. Tighten the cap so that the torque line on the cap passes the torque line on the vial.

4. Record the patient’s name on the vial and complete a cytology requisition slip including whether HPV or chlamydia/gonorrhea testing is desired, the date of the patient’s LMP, any hormonal therapy, a history of previous abnormal pap smears, and any pertinent treatments such as cryotherapy, conization, radiation, or chemotherapy.

Endocervical Brush/Spatula protocol:

1. Obtain an adequate sampling from the ectocervix using a plastic spatula.

2. 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.

3. Obtain an adequate sampling from the endocervix using an endocervical brush device. Insert the brush into the cervix until only the bottommost fibers are exposed. Slowly rotate ¼ or ½ turn in one direction. DO NOT OVER-ROTATE.

4. Rinse the brush as quickly as possible in the 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. Tighten the cap so that the torque line on the cap passes the torque line on the vial.

6. Record the patient’s name on the vial and complete a cytology requisition slip including whether HPV or chlamydia/gonorrhea testing is desired, the date of the patient’s LMP, any hormonal therapy, a history of previous abnormal pap smears, and any pertinent treatments such as cryotherapy, conization, radiation, or chemotherapy.

Autocyte thin-layer cytology (genital):

1. Insert the Rover’s Cervex-Brush into the endocervical canal. Apply gentle pressure until the bristles form against the cervix. Maintaining gentle pressure, hold the stem between the thumb and forefinger and rotate the brush five times in a clockwise direction.

2. Place your thumb against the back of the brush pad and disconnect the entire brush from the stem into the CytoRich preservative vial.

3. Place the cap on the vial and tighten.

4. Label the vial with the patient’s name and complete a cytology requisition slip including whether HPV or chlamydia/gonorrhea testing is desired, the date of the patient’s LMP, any hormonal therapy, a history of previous abnormal pap smears, and any pertinent treatments such as cryotherapy, conization, radiation, or chemotherapy.

Thin-layer cytology, nongenital:

Fluids such as urine, pleural fluid, peritoneal or ascitic fluid, bladder washings, or fluid obtained in the course of a fine needle aspiration should be placed in an equal amount of Cytology Fixative for Fluids. Large quantities of fluid (greater than 25-50 ml) may be sent fresh to the laboratory and a sample will be aliquoted and placed in fixative. If the fresh specimen may not be sent immediately, refrigerate until submitted. A monolayer slide will be prepared for microscopic examination.

Touch Preparation

Touch preparations are utilized to obtain a slide sample from solid tissue when either a smear is impossible to obtain (dry tap with a bone marrow aspiration), or when preservation of the relationship between the cells is desirable (i.e., lymph nodes, etc.).

A touch prep is made by touching wet tissue with a glass slide. This slide can be air dried (bone marrow) or immediately fixed (most other specimens) by immersion in 95% alcohol or with spray fixative. These should then be sent to PMA with the accompanying tissue. They will be processed as deemed necessary by the responsible pathologist.

Urine Cytology:

Specimen: 10-50ml fresh urine, bladder washings, ureteral washings, or renal pelvis washings placed in equal amount of Cytology Fixative for Fluids..

Collection:

1. Empty bladder upon arising in morning.

2. Hydrate by drinking several glasses of water ½ to 1 hours prior to collection.

3. Collect fresh specimen and place in Cytology Fixative for Fluids. Submit to the laboratory as soon as possible.

Washings (Bronchial, Esophageal, Ureteropelvic, Etc.)

Place washings for cytology in an equal part of “cytology fixative for fluids” (cytolyte). Label the container with the patient’s identifying information, specimen type, and source. Make sure the lid is securely tightened to prevent leakage. Place the specimen container in a biohazard transport bag with the completed requisition for submission to PMA.

Evaluation of washings is available Monday – Friday 8:00 – 5:00, and through special arrangement on the week-ends. Due to required processing and screening time, specimens received in the PMA laboratory after 2:00 p.m. will be processed the following day. Reports are generally available within 24 hours of receipt.