Specimen Collection Manual

Complete, accurate requisitions and correct specimen labeling are critical for ensuring positive patient identification, patient safety, and quality patient care.

• Complete all sections of the requisition.
• Label specimens with patient ID and appropriate specimen site/type. Place the label on the container itself and not on the lids.
• Place the specimen container and requisition/paperwork in a specimen transport biobag. The container goes in the main zip-lock part and the requisition in the outer side pocket.
• Incomplete information or specimen to requisition mismatch may result in a delay of results, the inability to process the specimen, or returning the specimen to your office.

A minimum of two (2) patient identifiers are required on the specimen container, one of which must be the patient’s name. Label as follows:

1. Patient’s full name (first and last, no nicknames).
2. A secondary, unique identifier such as date of birth, Social Security number, or other unique identifier of the patient.
3. Attach a peel-off barcode number from the requisition form to the specimen container. This label is unique to each requisition and aids in matching specimen container to their corresponding requisition.

For multiple specimen containers from the same patient and procedure:

1. Label with patient identifiers as above.
2. Indicate the specimen source and/or site on each container.

Important: The two patient identifiers used on specimen containers must match those on the requisitions. Failure to do so will result in the specimen being rejected and returned to the clinician.

Although PCNM has no enforcement of regulatory role or responsibility in your implementation of these regulations, we feel an obligation to assist our clients in ensuring that the specimens you send to PCNM meet the regulatory requirements.

PCNM will supply you with the proper packing and reference material to help ensure that the specimens are sent safely. PCNM provides the following guidelines as a service to our clients to assist properly trained shippers. This information does not take the place of formalized training.
If you are unsure about the regulations concerning shipping a patient specimen or have general questions about proper shipping methods and materials, please call our Logistics Coordinator.

Because they are in solutions that deactivate infectious organisms, the majority of patient specimens that you send PCNM are considered “exempt” from infectious materials regulations. They are, however, subject to the hazardous material transport regulations. Please call if you need to ship a specimen that is not in one of the fixative solutions.

When using the supplied materials, specimens must be packaged and labeled as follows.

The packaging must consist of:

• A leak-proof primary receptacle (the actual specimen container)
• A leak-proof secondary packaging (the plastic biobag)
• An outer packaging of adequate strength, with one surface at least 100 mm x 100 mm (4in x 4in) (the cardboard box)
• For liquids, absorbent material in sufficient quantity to absorb the entire contents must be placed between the primary receptacle and the secondary packaging. (use the proper size sheet of absorbent paper for the amount of liquid in the box)
• When multiple fragile primary receptacles are placed in a single secondary packaging, they must be either individually wrapped or separated to prevent contact between them.
• The outer packaging must be marked: “Exempt human specimen”

Requisition Required Information

• Performing clinician
• Collection date
• Patient name
• Patient Social Security number
• Patient date of birth
• Patient sex
• Pertinent clinical history or diagnostic question
• Specimen source and site
• Specific specimen information if there are multiple and/or different specimens from the same patient submitted on the same requisition
• ICD-10 code or symptom/diagnosis
• Insurance information (complete area on the requisition or attach copies of face sheet or card(s)
• Medicare patients: Attach signed ABN form to requisition

Requisitions and Supplies
Requisitions and supplies may be ordered by filling out the online Supply Request form, faxing the completed Supply Request Form to (575) 622-4795, or calling the Logistics/Supplies Coordinator at ext. 231.

Reporting
A completed surgical pathology report is usually available within one working day of receipt of the specimen.

Exceptions:

• Large specimens requiring overnight fixation
• Bone requiring decalcification
• Cases requiring special studies or additional sections
• Cases requiring consultative assistance

Rush handling of specimens is available. Prior consultation with the pathologist on-call is required for this service.

Information on cases delayed beyond 24 to 48 hours can be obtained by calling client services at ext. 203.

Patient requests for slides or results will be referred to the ordering clinician’s office. If the ordering clinician requests slides or results to be given directly to the patient, the patient must be physically present with photo ID to receive the slides or report.

Please Note: PCNM is NOT able to send out original slides, and must make recuts. Please allow time for recuts to be made. Exceptions to this rule may be made in certain instances. Sending of original slides requires consultation with the pathologist and their approval.

• Specimens should be submitted in an adequate amount of 10% neutral-buffered formalin fixative. The volume ratio of fixative to specimen size is very important for proper preservation of the tissue. As a minimum requirement, there should be at least twice the volume of fixative as tissue. The specimen should be placed in a container large enough to hold this amount of fixative. Small biopsies should be placed in at least 20 mL of formalin.
• For breast biopsies, be sure to indicate the time of the biopsy and the time the biopsy was placed in formalin. There is a place on the requisition for this information.
• Extremely large specimens (e.g. amputated limbs) should be placed in a large container or securely fastened plastic bag. If these specimens are not fixed, they must be double bagged, the outer being a red, appropriately labeled biohazard bag. Store the bagged specimen in a refrigerator until a courier can pick up.
• Questions arising outside of regular working hours are handled by the pathologist on- call.

Notes for Operating Room, Surgical Center, Ambulatory Care Personnel

• Ensure that the correct requisition form is properly filled out (e.g., Surgical Pathology for tissue biopsies and nongyn specimens; Gyn Pathology for gyn-related procedures). Indicate preoperative and postoperative diagnoses and clinical information as available.
• For breast biopsies, be sure to indicate the time of the biopsy and the time the biopsy was placed in formalin. There is a place on the requisition for this information.
• Submit properly labeled containers of appropriate size for the specimen and the correct amount of formalin.
• Generate a face sheet to accompany the specimen to the lab.
• For frozen sections during normal working hours, give the pathologist a 15-minute lead-time (in the case of a rural location, dates must be scheduled ahead of time). Be sure to contact the pathologist for any additional questions or when the situation is in any way unusual to you.
• Submit lymph node tissue fresh. Contact the pathologist on-call immediately for their handling of the specimen. If a pathologist can not be available or if there is a problem or delay, submit additional tissue in AZF fixative. Call the on-call pathologist for what to do if you do not have the AZF fixative, and how to handle these situations.

• Specimens should be submitted in an adequate amount of 10% neutral-buffered formalin fixative. The volume ratio of fixative to specimen size is very important for proper preservation of the tissue. As a minimum requirement, there should be at least twice the volume of fixative as tissue. The specimen should be placed in a container large enough to hold this amount of fixative. Small biopsies should be placed in at least 20 mL of formalin.
• For breast biopsies, be sure to indicate the time of the biopsy and the time the biopsy was placed in formalin. There is a place on the requisition for this information.

• Appointments should be made in advance for frozen sections by calling PCNM between 5:00 am and 5:00 pm (575) 622-5600 or (800) 753-7284 ext. 218. The pathologist on-call should be contacted at least 30 minutes in advance of the frozen section on weekends, holidays, or outside regular working hours. Frozen sections, intraoperative consultation, and FNAs other than at our full-coverage institutions are to be scheduled in advance and on scheduled visit days.
  
• Tissue for frozen section must be delivered in the fresh state with care taken to avoid the specimen drying out during transport. A completed Surgical Pathology requisition and patient face sheet must accompany the specimen.
• Upon completion of the frozen section, the pathologist will verbally contact the surgeon with the diagnosis.

• All diagnostic lymph node biopsies should be promptly evaluated by a pathologist in the fresh state to ensure that the proper studies are performed. The on-call pathologist should be contacted to handle the specimen. If an infectious process is suspected, additional sterile tissue should be sent directly to the microbiology laboratory at your institution for culture.
• Lymph nodes removed in a physician’s office and for a suspected diagnosis of possible lymphoma should be wrapped in saline-moistened gauze and PCNM should be contacted immediately for pick up (575) 622-5600, ext. 218.

Sentinel lymph nodes are identified using a supravital dye and/or radioisotopes. IHC cytokeratin staining will be performed routinely if negative by H&E.

Sentinel nodes that were identified via radioisotopes will be placed in formalin (if not already) and might be stored for 24 hours prior to handling to allow for radiolabel decay.

The most important step is having prior notification from the surgeon that he/she is going to perform a muscle biopsy.

Preferably, we should have at least 2 to 3 days prior notification. It is also important that biopsies not be obtained on a Friday as the tissue may deteriorate over a weekend.

• The submitting surgeon must supply PCNM with sufficient history, physical examination findings, appropriate laboratory studies, and ancillary tests (e.g., EMG findings or others).
• Obtain the biopsy from the most appropriate site. It is recommended that the surgeon does not inject Novocain into the biopsy site or cauterize.
  • It is important to obtain the biopsy from the muscle belly, not adjacent fascia
  • Obtain good longitudinal fibers; do not dissect using cautery as this creates significant artifacts. Obtain 5 to 6 mm x 2.5 cm of tissue.
• Once the tissue is obtained, wrap the tissue in saline-soaked gauze. Do not immerse the skeletal tissue into saline. It is important that the tissue does not dry out. DO NOT allow the tissue to arrive floating in normal saline because the enzymes that are tested for are water soluble.
• Have the appropriate operating room (OR) personnel contact PCNM to pick up the tissue.

Renal biopsies may be done with a Tru-Cut needle or a biopsy gun; with the latter, the specimens are much thinner, so a minimum of two cores should be obtained. The biopsy(s) should be received fresh, accompanied by appropriate clinical information.

1. Two separate cores: Place one core in formalin for light and electron microscopy (LM and EM) studies. Place one core in Michel’s fixative for immunofluorescence (IF) studies.
2. Single core/scant material: The core should either be divided in half for light and immunofluorescence or submitted entirely for light microscopy.

One sample should be in 10% neutral-buffered formalin, and the other in Michel’s solution (supplies available through PCNM). Be sure the Michel’s is not outdated. Call PCNM with any questions. If the specimen is from an uninvolved site, please state such on the requisition.

• Tissues suspected of being involved with a primary or metastatic breast carcinoma should be submitted for ER/PR/HER2/neu (Herceptest) Assay and other predicative markers.
• Time without and with fixation affect the quality of the predictive markers. It is essential to indicate the time of the biopsy and the time the biopsy was placed in formalin. There is a place on the requisition for this information. Please ensure these specimens are picked up immediately, and that they reach the lab no later than 48 hours post collection to comply with CAP guidelines for breast tumor markers.
• Analysis will be performed on fixed, paraffin-embedded section.

Peripheral Smear(s) and CBC

• Preferably on fresh drawn blood from the day of the procedure, or from a day or two prior to the procedure.
• At least four slides should be made. If your facility has an approved Wright-Giemsa staining method, two of the slides should be stained and two left unstained.

Bone Marrow Aspirate and Smear(s)

• From the fluid drawn, several (10 to 12) smears should be prepared and allowed to air dry. Aspirate smears are thicker and should have particles present. These will appear as translucent shiny dots in the central portion of the slides and are the actual bone marrow particles.
• If your facility has an approved Wright-Giemsa staining method, two of the slides should be stained and the remainder should be left unstained.
• A portion of the aspirate should be submitted in two green-topped blood vials for flow cytometric and cytogenetic studies.
  The remainder of the aspirate material is allowed to clot and be placed into formalin.

Bone Marrow Core and Touch Preparations

• From the needle-core biopsy, imprint/touch prep slides are made (8 to 10).
• If your facility has an approved Wright-Giemsa staining method, two of the slides should be stained and the remainder should be left unstained.
• Once the slides have been prepared, the biopsy can be placed in formalin.

Additional Protocol for Bone Marrows

• All slides, stained and unstained, should be forwarded with the formalin-fixed aspirate clot and core specimens to PCNM.
• The aspirate specimens for flow cytometry and cytogenetics must be forwarded by your facility directly for optimal testing results.
• Do not send the aspirate blood for flow and cytogenetic testing to PCNM. Contact PCNM for procedure.
• A copy of the CBC values of the patients’ blood on the same day the bone marrow was collected is also required for interpretation. If this is inadvertently not done, the nearest CBC values to the date of the bone marrow procedure should be submitted.
• All slides should have two identifiers, one of which must be the patient’s name and the other a secondary, unique identifier (e.g., date of birth or Social Security number). The remainder of the specimens also require a minimum of two patient identifiers, one of which the patient’s name. They should be submitted with a completed PCNM requisition, Bone Marrow Examination form, and CBC report.

• Remember that all specimens should be considered infectious and handled according to Universal Precautions.
• All specimens should be placed in sealed biohazard bags prior to transport.
• Fresh specimens are preferred. Never add formalin, absolute alcohol, or other fixatives to a cytology specimen. A 1:1 mix of CytoLyt:Specimen volume can be used if delay in transport is anticipated. Refrigeration is preferred over fixative. Please call us to verify the need for CytoLyt before adding it to any specimens.
• Check that all containers with screw caps are tightened securely to prevent leakage. CytoLyt, PreservCyt, and ThinPrep Pap vials have lines on them to help ensure that they are securely tightened. Tighten these so that the mark on the cap passes the line on the vial.
• When syringes are used to obtain cytopathology specimens, needles must be removed and replaced with syringe caps.
• Flow cytometry specimens should be submitted in RPMI.
• All specimen containers and slides must be properly labeled:
  • With the patient’s name and a secondary identifier (such as date of birth, Social Security number, or other identifier unique to the patient).
  • Specimen site/type. Be sure to indicate if left or right and/or any other specimen site info, especially if more than one site is being sampled.
  • Label the container itself and not the lid.
  • And accompanied by a properly completed requisition that has all pertinent patient, specimen, clinical, and billing information.
• Mismatch of patient identification between the specimen and requisition will result in the specimen being rejected.
• For complete specimen submission instructions see Specimen Labeling and Handling – General Information

Labeling Instructions: Pre-label frosted end of glass slide with patient’s full name and whether the specimen is from the left or right breast.

Collection-Smear on glass slide and fixative (preferred method):

1. Wipe the surface of the nipple with the gauze square to remove anucleated cells & debris.
2. Gently massage subareolar area and nipple with thumb and forefinger.
3. When secretion occurs, allow a pea-sized drop to accumulate. Touch the secretion with a clean glass slide and withdraw quickly.
4. Immediately, spray the slides with 3-4 sprays of the cytology fixative in order to preserve the cells. Time is of the essence for good preservation – don’t let the slide dry before applying the fixative.
5. Repeat procedure until all secretions from the nipple are collected. As many slides as possible should be attempted due to the fact that diagnostic cells most often appear on the latter slides and that the first few slides often contain degenerated cells.

Special Instructions: If the nipple secretion is thick, make additional slides using the “pull-a-part” method. Hold a second slide gently on top of the first, allow weight of the slide to spread the specimen, and then quickly pull the slides apart. Fix each of the slides immediately before specimen dries.

Fine-needle aspiration (FNA) with air-dried smears / wet-fixed smears:

1. Label slides and CytoLyt vial with the patient’s name and date of birth. Also label with the specimen site. Indicate if left or right or other relevant specimen site info, especially if more than one site is being sampled.
2. Take one pass and express one or two drops from needle at middle of slide.
3. Place another slide over the drop and allow the drop to spread.
4. Separate the two slides. Spray cytology fixative to one slide immediately and allow the other slide to air dry. Verify the labeling of the slides.
5. Make at least three to six passes trying to sample the mass thoroughly and express the specimen into the CytoLyt solution tube.
6. Rinse the needle by drawing approximately 5 mL of the CytoLyt fluid into the syringe and re-express back into the tube.
7. Repeat this with each needle and syringe used.
8. Check that the labeling of the CytoLyt tube and slides have been completed appropriately.
9. Send these to the lab along with all pertinent clinical information including level of clinical suspicion for malignant lesions and radiographic findings.

Collection Supplies
A. Preferred Collection Method in Cytology Collection Fluid:

• Dacron swab (not cotton, as cotton tends to trap and hold cells), or cytobrush
• PreservCyt or ThinPrep Pap vial of collection solution
• View the Anal Cytology Quick Reference Guide PDF

B. Alternative Sample Collection Method—Smear and Glass Slide

• Dacron swab or cytobrush
• Glass microscope slides for making smears
• Cytology fixative Spray

Be sure to use the appropriate collection media for the intended testing. If the test collection media you have is not listed below, please call for guidance prior to collecting the specimen.

PCNM tests include, HPV, CT/NG, Vaginosis panels. Collection media options vary with each test.

From the ThinPrep vial we can test for:

• HPV
• CT/NG
• Extended vaginosis panel
• HSV
• Trichomonas

Manufacturer Test Kit must be used for:

1. CT/NG testing with Gen-Probe Aptima Combo 2 test kits:
   • Unisex Swab Kit: endocervical swab and urethral male
   • Urine test kit for females and males
2. BV/CV/TV Panels