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LABORATORY OF PATHOLOGY ONLINE POLICY MANUAL

Specimen Requirements, Handling, and Incident Management

Histopathology Section

PURPOSE
The specimen management procedures in the Histopathology laboratory are intended to support the research mission at The National Institutes of Health while also preserving samples for clinical diagnosis. The procedures that follow are intended for the Histopathology laboratory, but there are other procedures that involve specimen handling in the operating rooms and procurement laboratories. For a more detailed description of procurement procedures, refer to the Specimen Collection Guide and Surgical Pathology Rotation procedures at http://home.ccr.cancer.gov/lop/intranet/policymanual/default.asp

PRINCIPLE
The National Institutes of Health is a research institution and as such, it is common to have specimens removed from the Operation Rooms (OR) without fixative. This practice allows for sampling fresh tissue for research studies that are not possible on chemically fixed tissues. As a consequence of this mission, the Histology laboratory receives specimen that may be fresh or in fixative. The laboratory also receives specimen from clinics throughout the building and consult cases that usually consist of paraffin blocks and glass slides.

COMMONLY USED TERMS
The following terms are frequently used in this procedure:

  • Specimen: any product of a medical procedure. These can be soft tissues, boney tissue; fluids, foreign bodies, surgical appliances/hardware. The terms “sample”, “specimen”, and “tissue” are often used interchangeably.

  • Fixative: a solution used to stabilize cellular components in preparation for histological examination. Proper fixation is essential for histology, but it kills cells and acts in other ways that limits many research applications. 10% neutral buffered formalin is the most common routine fixative.

  • Fresh: no fixative has been used. Fresh tissue samples must be frozen, placed in a transport media to keep the cells alive, or stabilized in some other way as soon as possible to prevent autolysis.

  • Frozens: specimens that are or have been frozen for rapid microscopic exam during an interoperative consultation. Frozen section slides are cut on a cryostat for rapid microscopic analysis. The remaining tissue is placed in fixative to be made into permanents.

  • Permanents: specimen that have been treated with fixative and processed to a paraffin matrix that permanently preserves them at ambient temperature. “Paraffin block” is an equivalent term. “Permanents”, or paraffin blocks, are specimen for which a microscopic histological exam can be performed. The histology of permanents is superior to that of frozens.

  • Gross only examination: a descriptive exam of the macroscopic features of the specimen that does not include a microscopic exam. Specimens for “gross only” are usually foreign bodies or otherwise not practical to be made into permanents.

  • Procure: the process of allocating portions of the specimen to fulfill the research protocol requirements while preserving representative portions for clinical diagnosis.

SUPPLIES

  • 10% neutral buffered (NBF) in prefilled specimen containers (SurgiPath #00790)

  • 10% neutral buffered (NBF) in 20L cube containers (Scientific Supply #EKI-4499-20L)

  • HistoChoice fixative, supplied by the investigator

  • Plastic containers, non-sterile, various sizes, no particular vendor requirement (example: Cardinal Health #ES3704)

  • 10% formalin hazard label (EMS #77060-16)

  • Specimen refrigerator set 0 to 8 degree C. The colder setting is to counteract the effect of large, warm samples.

SPECIMEN ACCEPTABILITY IN THE HISTOPATHOLOGY LABORATORY
The attending pathologist, in collaboration with the grossing resident, directs the handling of each specimen on a case-by-case basis. A general guideline is as follows:

The following ARE ACCEPTABLE specimens for pathological evaluation:

  • OR specimens, previously procured, fixed in 10% formalin

  • OR specimen, previously procured, fresh. Smaller fresh specimens should be kept moist in saline or a transport media such as RPMI.

  • Frozen Section specimens, consultation completed, in 10% formalin

  • Clinic biopsies fixed in 10% formalin

  • Hematopathology lymph nodes, previously procured in Hemtopathology, in 10% formalin

  • Autopsy cassettes, labeled with the autopsy number, in 10% formalin

  • Breast protocol specimens in HistoChoice fixative

  • Any foreign bodies such a ports, stents, etc. for which a Surgical Pathology CRIS order has been received. In general, these will be handled as “gross only” cases for which no samples are submitted for microscopic evaluation. These can be submitted in dry specimen containers. If there is adherent tissue, that material may be submitted for paraffin processing at the discretion of the resident and attending. These should be submitted in formalin to prevent decay, even if the tissue will not be processed for examination.

  • Consult materials consisting of fixed wet tissue, paraffin blocks, and/or glass slides

  • Frozen material if requested by an attending Laboratory of Pathology (LP) pathologist only.

  • Teeth. Usually these will be for gross examination only, but adherent tissue may be submitted for microscopic examination at the attending’s discretion.

The following are generally NOT acceptable specimen unless otherwise directed by the attending pathologist:

  • Bone marrow cores. These go to the Clinical Pathology lab

  • Ocular specimens. These go to the Ocular Lab.

  • Any specimen fixed in gluteraldehyde. These go to the Electron Microscopy (EM) lab unless specifically requested for paraffin processing by an attending LP pathologist only.

  • Any fresh specimen for which flow cytometry or cytogenetics samples must be taken. These must be brought to the attention of the resident or attending immediately and before any fixative is added.

  • Blood and other fluids [such as bronchoalveolar lavage (BAL), pleural, peritoneal, cerebrospinal (CSF), sputum] and smears made from these. These should go to the Clinical Pathology lab or Cytology, depending upon the sample and test requested.

  • Frozen aliquots of anything (see the exception above)

  • Radioactive specimens are not expected. If anything is received that is marked as radioactive, place in a refrigerator and contact the Director of Clinical Operations immediately. See also the LP on-line policies.

TRANSPORTATION

All fresh specimens received from the Operating Room should be received in Room 2C533 with the proper identification labels.

Wet specimens are transported to the Histopathology laboratory by Escort Services, clinic staff, or LP staff. General institutional policies for specimen transportation apply and include that containers are properly closed to prevent leaks. Secondary containers such as specimen bags and/or transport buckets marked with the biohazard symbol are used. Containers that contain fixative are labeled as such. All transportation can be done at room temperature. Fresh samples must be transported to refrigeration without delay. If this is not possible, transport in a container on wet ice so as to keep the sample cold, but without contaminating the sample with the ice.

Samples brought by Escort Services must be logged into the log book at the laboratory entrance. This documents the time of delivery and allows laboratory staff to identify samples that are mis-delivered (see Specimens Acceptability, above).

No infectious, frozen or wet tissue is routinely shipped to off-site locations. If shipping is required, it must be done by staff that has current training in IATA Dangerous Goods Regulations.

SPECIMEN PRESERVATION

  • Specimens in 10% formalin: Anything received in 10% formalin can be held at room temperature until grossed by the resident. Refrigeration (2-8 degree C) is not needed and should be avoided so as not to slow down the chemical reaction. These specimens may be received in pre-filled formalin containers, which the Histopathology laboratory provides, or in generic specimen cups to which 10% formalin has been added. The later must be labeled with a 10% formalin hazard label. If the volume of fixative does not cover the sample, or the sample is squeezed into a container that does not allow enough formalin, contact the resident to add more formalin or transfer to a larger container. The volume of fixative should be at least two times that of the specimen.

  • Specimens received fresh: Anything received fresh (without a fixative such as formalin or alcohol) must be brought to the attention of the resident to ensure proper handling. In the interim, fresh specimens must be refrigerated (0-8 degrees C) until the resident makes sure the sample has been procured. Large samples from the OR must be covered with Parafilm or similar material. Smaller samples should be kept moist with a Tyvec or similar pad moistened with saline or RPMI and submitted in a sealed specimen container.

FIXATION TIMES
In general, biopsies are submitted for processing to permanents the same day as received if they were received in formalin. Larger specimens may or may not be cut into cassettes the same day as received depending upon the tissue type and how long it has been in formalin. Some specimen types have specific handling requirements to optimize fixation times as follows:

  • Breast: Since these specimen may require Her2 testing, their fixation must be no less than 6 hours and no more than 48 hours per CAP #ANP.22998. As a consequence, there are special procedures for samples that come in at the end of the week or before a Holiday. See the grossing procedures.

  • Brain, larger than a biopsy: Since brain is particularly soft and formalin penetrates slowly, the grossing procedure is designed to cut the tissue into cassettes as quickly as possible and allow them to fix overnight before they are submitted for processing. See the grossing procedures.

  • Hematopathology lymph nodes: at the direction of the hematopathologist, most larger nodes are cut into cassettes and allowed to fix overnight before they are submitted for processing.

  • Radical prostatectomies: The glands are allowed to fix overnight before being breadloaved the following working day. The breadloaves are allowed to fix an additional day before processing. See the grossing and prostate procedures.

CRIS REQUISITION and LABELING REQUIREMENTS

  1. CRIS Requisitions: All cases must be accompanied by a requisition form (CRIS). Verbal test orders are not accepted. There must be one CRIS requisition per patient and per procedure. The only exception is specimens from an on-going surgical procedure. It is expected that the CRIS is generated as soon after completion of the surgery as possible and no later than 0900 hours the following morning. This requisition is electronic and will include essential information such as the CRIS number (which is used to accession the case in the LIS); patient demographics; collection date; specimen description; the submitting physician’s name; and other physician contact information. Significant clinical data/diagnosis is expected, but does not constitute a specimen deficiency if not provided. The form itself prints from two printers, one in the Histopathogy lab and one in the Frozen Section room. Only cases for Surgical Pathology print at these locations and this defines the type of examination requested. If the CRIS is not on either printer and not with the specimen(s), it is considered missing.

  2. Container labels: All specimen containers must be legibly labeled with the following:

    • At least two (2) patient identifiers. The first identifier must be the patient name and the second may be the medical record number (MRN) and/or date of birth (DOB). A computer printed patient demographic label is preferred whenever possible.

    • The anatomic source of each specimen on each container.

    • A fixative label, once the sample is in fixative. Most often this will be a 10% formalin hazard label.

  3. CRIS/Specimen agreement: The patient identifier labeling on the specimen containers must match that on the corresponding CRIS. The anatomic source descriptions between the specimen containers and the CRIS will usually not match word-for-word, but the descriptions must be consistent. The resident or attending pathologist must be consulted if there is a question about matching source descriptions. During accessioning, the CRIS and all associated specimen containers are compared for proper labeling. When either the specimen container label or the CRIS is missing or mislabeled, some corrective action is required. See Specimen Problems below.

  4. Pathology case number: At accessioning, a pathology number is assigned to the patient sample. That pathology number becomes the unique identifier for that patient, sample and date of procedure. The pathology number is placed on the CRIS and any other paperwork, all specimen containers, tissue cassettes, and slides. It therefore becomes the link to identify the patient and every sample taken from that patient per date of service. See the SoftPath procedures for accessioning instructions.

SPECIMEN and CRIS REQUISITION PROBLEMS

Problems include: no CRIS requisition received; specimen container with no label; mismatching demographics; mismatching specimen source between the CRIS and one or more specimen containers; no tissue identified; etc. In general, anatomic pathology specimens are not rejected for testing because they are irreplaceable and/or cannot be easily recollected. However, any problems must be documented and corrected as the grossing resident, attending pathologist, and/or the Director of Clinical Operations see appropriate.

  1. Missing CRIS orders: Verbal test orders are not accepted and it is not possible to accession cases until the CRIS number is received. Therefore missing CRIS orders should be handled as follows:

    • The grossing resident and Histology staff will work together to identify cases with missing CRIS orders. The resident must page the appropriate staff to get missing CRIS orders as follows. Note: make sure the responsible staff entering the CRIS know to designate the order for Surgical Pathology.

    • OR samples: page the Fellow listed on the OR schedule or page the On Call Surgery Fellow

    • TIL samples: page the On Call Immunotherapy Fellow

    • Urology (UOB) samples: page Dr. Bratslavsky, Dr. Pinto, or the On Call UOB Fellow.

    • Heme/Onc samples: page the hematopathologist or her staff so they can contact the relevant parties.

    • Clinic specimen: these are dropped off to the Histopathology lab by Escort Services and they cannot help with CRIS problems. Look up the patient in CRIS via the MRN. Contact the primary physician listed.

    • If the physicians cannot be reached or they do not generate a CRIS order, the resident must notify the Director of Clinical Operations. Histopathology cannot discard a specimen once it is in the laboratory unless approved by the Director, but the specimen can be returned to the clinic/OR if repeated attempts to get a CRIS fail.

    • When the CRIS is received, the Histology staff will accession as usual, but the delayed CRIS problem will be documented in SoftPath as specified in the SoftPath procedures.

  2. Mislabeled or unlabeled specimens:

    1. For practical considerations, if a case consists of one specimen container, and it is otherwise complete except for the anatomic source on the specimen container, the case is not held for verification. The Histology staff will accession using the specimen source listed on the CRIS and flag the labeling problem in SoftPath. The grossing resident should include in the dictation that the specimen container was labeled with the patient’s name, but not with an anatomic source.

    2. All other labeling problems should be handled as follows:

      • The Histology staff will hold the specimen (s) aside without accessioning it and notify the grossing resident and/or attending pathologist immediately. Keep unfixed samples refrigerated.

      • The grossing resident must contact the service that generated the sample. Use the same contact list as for a missing CRIS orders (see above). Most problems like left side/right side discrepancies will require that the surgeon, clinic physician, or designee come down to the Histology lab to fix the error and initial the correction. Specimens with the wrong or missing patient label must be corrected with new patient labels supplied by the service that generated the sample.

      • If the physician cannot be reached or they do not correct the problem, the resident must notify the Director of Clinical Operations. If the specimen is not fixed, the Director may opt to place in 10% formalin, even if it is unclear what testing was intended.

      • When the labeling is fixed, the Histology staff will accession as usual and flag the problem in SoftPath as specified in the SoftPath procedures.

  3. Specimen Quality Problems: Such problems include inappropriate fixation; delayed fixation; delayed delivery; leaking container; desiccation; no tissue seen in the specimen container; badly damaged blocks; shattered slides; etc. Receipt of such specimen does not necessarily preclude its histologic examination, but may limit its suitability for ancillary studies and diminish its clinical utility. Such problems should be handled as follows:
    • The Histology staff will notify the grossing resident and/or attending pathologist immediately. Keep unfixed samples refrigerated.
    • The pathologist, together with the attending and /or Director of Clinical Operations will be responsible for determining how the specimen should be further processed. In all cases, the requesting physician should be notified of the problem. If applicable, Escort Services (301-496-9295) must be contacted and educated so as to avoid repeat errors.
    • If the pathologist wants to proceed with case, the Histology staff will accession as usual and flag the problem in SoftPath as specified in the SoftPath procedures.
    • The resident must document the specimen condition as it was received and what corrective action was taken. This should also be dictated with the case so as to become part of the medical record. If no tissue is present in the specimen container, the specimen is dictated as “no tissue received in the container.”
    • Unclear test orders: The Histology staff will notify the grossing resident and/or attending pathologist immediately. The pathologist will be responsible for clarifying any unclear orders, including contacting the service that generated the sample, if needed. The resident must document the corrective action taken and may include this in the case dictation. Use the same contact list as for a missing CRIS orders (see above).

INCIDENT REPORTING
As indicated in this procedure, many problems can be flagged in SoftPath by the person accessioning the case. These flags are captured and reviewed by the QM Committee so as to identify trends that can be corrected.

Any problems that cannot be adequately captured in SoftPath must be brought to the attention of the Histology supervisor. These will generally be labeling and specimen quality problems. The supervisor is responsible for reporting such problems using the Quality Improvement and Assessment Log. The incident report must be submitted to the Clinical Laboratory Manager as indicated in the QA/PI Problem and Variance Report procedure.

SPECIMEN RETENTION TIMES
Empty specimen containers are held in the event that the demographics need to be checked. These contain no remnant tissue or formalin. They are kept in burn boxes labeled with the resident’s name and the date they can be discarded. The boxes are held for one month after the resident’s rotation is complete.

Specimen containers with remnant tissue in fixative are stored in the Histology lab for the month that the resident is on grossing rotation. They are transferred to the Autopsy suite when they are kept for at least another month before being discarded.

Paraffin blocks and glass slides are not discarded. CRIS orders and associated accession paperwork are kept for two years.

Last Updated 12/28/2009 1:16:59 PM

 
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