Overview

The aim of this manual is to complement the training of pathologists, registrars and non-pathologist staff in the process of macroscopic cut-up.1 Information is provided for the majority of specimens likely to be received in diagnostic histopathology laboratories. It is intended to be advisory and not prescriptive in nature. Therefore specific protocols in your laboratory may vary from the details provided here. However, the instructions reflect well-established cut-up methods, edited and reviewed by pathologists with considerable expertise in relevant disciplines. See project development and contributors for more information.

It is recognised that lab personnel with varying levels of knowledge and experience will access this information. Therefore in the first instance basic outlines on each specimen are provided, with further detail available by selecting "More detail" throughout the text. On the right, "View all" will open all hidden detail on the page.

How to use the manual

The manual is organised by organ systems and specimens can be found via the cascading menu or the search field on the left of each page. Within instructions for a particular specimen (e.g. uterus) there may be alternatives for different clinical situations (i.e. cervical intraepithelial neoplasia v endometrial neoplasia).

Cut-up instructions for each specimen follow this general chronological order:

  1. Background
  2. External inspection
  3. Dissection (if required)
  4. Internal inspection (if required)
  5. Processing
  6. Block allocation key
  • Headings with information to be recorded at each step are provided.
  • Each specimen page is presented in diagrammatic and dot point format with links to more detailed instructions, images, videos and references. The branching hierarchy can be followed to the level of instruction needed in each circumstance.
  • "Jump to" links are available on the right-hand side to move quickly to sections lower down the page.
  • Photographs and diagrams will enlarge when selected.
  • The website is mobile-device responsive allowing access on computer tablets and mobile phones.

Essentials of macroscopic cut-up

Cut-up supervision

Specimens received in Anatomical Pathology laboratories will require differing levels of cut-up expertise according to the complexity of the tissue. However, it is essential that all specimens are handled to ensure optimal microscopic examination can be undertaken by the reporting Pathologist.

  • Although cut-up may be undertaken by staff other than Pathologists, training and supervision must be provided and competency demonstrated in accordance with standards established in the Australian accreditation process. 1-5
  • It should be noted that, “the Pathologist has the ultimate responsibility for the handling of a Specimen regardless of who has performed the Cut-up”. 1,4
  • It is strongly recommended that users of this manual have a thorough understanding of standards and guidelines 1-5 relevant to specimen cut-up.
Structured reporting

Information in the manual is designed to support structured reporting rather than narrative descriptions. Reporting templates may be established from the minimum data items listed for each specimen.

  • Dictation templates are provided as downloadable pdfs to assist with this process.
  • Structured Pathology Reporting of Cancer (SPR) macroscopic reporting data elements are included in the dictation templates, aligned with SPR terminology and including SPR references numbers for standards and guidelines.
  • Links to the SPR website and other "related items" are provided below the left hand menu.

International units of measurements (SI) are recommended and it is standard practice in Australia to measure in millimetres not centimetres.

Identification

Labelling errors are infrequent but can have a significant impact if undetected. 6,7 It is a requirement (under Australian Standards) to ensure traceability of the specimen throughout all stages of processing in a medical laboratory. 2 Three identifiers must be used on the request form and the specimen container. 8,9 Best practice includes at least two identifiers on cassettes and slides labelled in the laboratory with correlation at each point of transfer (from specimen container to cassette, cassette to slide and slide to report). Correct identification can then be assured from receipt of the specimen through to reporting.

  • Three identifiers commonly used on the specimen container and accompanying request slip are the patient's full name, date of birth and unique record (UR) number/laboratory information system (LIS) identification number.
  • Two identifiers on cassettes and slides are usually the lab accession number and patient's surname.
Specimen transfer and contamination

Laboratory staff should be alert at all times to the risk of specimen transposition during cut-up and processing. Transposition error can result in a false positive or false negative diagnosis which, if undetected, can cause substantial patient harm.

  • Transfer of tissue to cassettes should be undertaken in an environment free from distraction
  • Visual correlation should be made that the label on the specimen container matches with the request slip and the processing cassettes.
  • During cut-up only one specimen container should be open on the bench at any one time. Once tissue is transferred to cassettes, lids should be closed prior to commencing on the next patient's specimen.
  • Cross over contamination is a potential source of diagnostic error 10,11 and should be considered as a risk at every stage of specimen handling. 12
  • Forceps, scalpel blades, knives, cut up surfaces, ink dyes and processing solutions can transfer tissue cells and fragments from patient to patient. 10-12
Clinical information

Appropriate clinical information must be provided by the referring clinician 13 and reviewed before commencing cut-up. Cut-up methods may vary dependent on the suspected pathology.

  • Information on specimen containers such as the clinical history, procedure, tissue type and biopsy site should be recorded as stated by the clinician and compared with the request description. Clarification of ambiguities should be sought from more senior staff or the referring clinician to ensure correct handling of the specimen.
  • The type of fixative present must be checked before cut-up commences. Specimens received fresh or in saline may require special studies such as microbiology, cytogenetics, flow cytometry, immunofluorescence and/or tissue banking. See also RCPA guidelines on biobanking. 14 Samples should only be taken for other purposes where there is sufficient tissue to ensure accurate histological examination. Record the details of any samples taken in the macroscopic description.
  • Check the specimen received matches the clinical description.
  • Note the receipt of all fresh specimens.
  • Describe the type and results of all intraoperative procedures undertaken on the specimen.
  • Ensure samples for other tests are taken if required and record the details.
  • Check if bone is present and decalcify after sufficient fixation (see decalcification for more detail).
  • Initials of staff involved in cut-up are usually recorded in the report for traceability of the specimen from reception to processing.
  • Structured Pathology Reporting of Cancer guides provide more detail on clinical information reporting requirements.
Tissues for other tests

Take great care with specimens required for other tests such as microbiology, cytogenetics and cytology. Specimens may require sampling by the other laboratory before histology processing can proceed.

  • Consult laboratory procedures and protocols before processing the specimen.
  • Avoid delays in processing fresh specimens.
  • Use an aseptic technique to divide or sample the specimen.
  • Ensure a sterile environment for division of the specimen.
  • Avoid contamination with other sources of DNA and RNA.
Occupation Health and Safety

Occupational health and safety (OHS) must be considered at all times in the histology laboratory.

  • Personal protective equipment must be used to prevent physical injury, exposure to chemical agents and transmission of infectious disease.
  • While all specimens should be treated as potentially infectious, fresh unfixed tissue is of particular risk and should be handled by experienced staff in appropriate extraction cabinets.
  • Specimens from known (or suspected) Human immunodeficiency virus (HIV), Hepatitis B (HBV) , Hepatitis C (HCV) and tuberculosis (TB) patients should be treated with extreme caution. 14,15
  • Avoid procedures that produce aerosols when handling unfixed tissue (e.g. use of bone-saws, pressurised sprays in the cryostat). 14
  • Note that mycobacteria has reduced infectivity but may survive formalin fixation. 15
  • Potential Creutzfeld-Jakob disease (CJD) specimens require special treatment as prions are not inactivated by formalin fixation in isolation. 15,16
  • Protocols for decontamination procedures must be in place with particular consideration of equipment used for fresh tissue such as cryostats. 17
‚ÄčSee also fixation for OHS in regard to formalin.
External inspection

Orientation of the specimen may be made from clinical notes, surgical sutures and anatomical features.

  • Laterality should be confirmed wherever possible against clinical notes and labels.
  • Adherence to strict quality assurance measures is recommended to prevent the mislabelling of specimens.
Description

Record the surgical procedure or specimen type as stated by the clinician on the specimen container and/or request form. Information on specimen integrity such as whether opened or disrupted and the number of pieces received should be noted if applicable (recording "intact" or "unopened" is optional).

  • Generally three dimensions of the specimen are recorded (in mm). Occasionally, only two dimensions are relevant such as length and diameter. Small specimens may only require measurement of the maximum dimension. Weight (in g) is recorded in some instances.
  • The general appearance of the external surface is described where necessary.
  • Important features are recorded such as the presence of perforation, adhesions, foreign bodies and evidence of previous surgery.
  • The colour and texture of a specimen is not always relevant to staging, prognosis or microscopic interpretation and may not require description unless indicated in specific tissue protocols.
  • Photographs are a valuable record of the specimen’s macroscopic appearance.
Dissection

Paint relevant margins with ink if necessary, following your laboratory’s protocols. Open the specimen according to its particular anatomy.

  • Directions, diagrams and photographs are provided throughout the manual as a guide to margin inking, dissection and block selection.
  • After opening, specimens will generally require longer fixation in larger quantity of formalin to ensure adequate preservation of tissue. 18,19 See fixation for more information.
Internal inspection

The internal or cut surface should be examined for the presence of lesions and abnormalities.

  • Lesions should be described with an emphasis on the maximum size, distances from margins and impact on other structures in the specimen, particularly for cancer staging purposes.
  • The general appearance such as colour, consistency and configuration may be relevant.
  • Photographs of dissected specimens can assist in recording the appearance of the disease process for the reporting pathologist.
  • A record of all photographs taken, diagrams recorded and markings used for identification should be included in the macroscopic description.
Processing

Further dissection into thin slices (3-4mm thick) will ensure optimal processing. Submit sufficient tissue for processing to demonstrate the required features of the particular specimen. Completeness of excision is an important item in histopathology reports. Therefore the blocks selected must demonstrate the relationship of lesions to adjacent normal tissue and margins. Tissue sections should fit comfortably within the processing cassette.

  • Ensure that the specimen is adequately fixed before processing and use an appropriate processing schedule for the type of specimen.
  • Overcrowded cassettes and thick tissue sections will not allow adequate penetration of solutions and wax during processing, resulting in poor microtomy.
  • Prevent the loss of tissue during processing with the use of suitable cassette liners.
  • Commonly laboratories utilise commercially available foam sponges, biopsy bags, lens paper or filter paper liners for small biopsy and friable specimens. Some institutions include liners in every cassette to minimise cross over contamination during processing. 12 Paper is more suitable than sponges for larger specimens (as it does not significantly reduce the volume of the cassette).
Block allocation key

A record of the block selection should be included in the macroscopic description which records each cassette’s labelling, the site of sections submitted and the number of tissue pieces processed. For illustrative purposes alphabetical labelling has been used in this manual but a variety of alphanumeric labels are possible. The number of pieces submitted for processing is correlated against number of pieces seen microscopically so the description must be clear and accurate. 20

  • Annotated photographs of the dissected specimen, indicating the blocks processed, are a useful block allocation key.
  • Dissection diagrams with recommended areas for block sampling are provided throughout the manual. Tables are also provided as examples of block allocation keys (as below). The fields for the number of tissue pieces are blank but are intended as a prompt to record this information in the macroscopic description.

Cassette id

Site

No. of pieces (per cassette)

 

 

 

 

 

 

 

 

 

Specimen retention

Remaining tissue should be stored for a sufficient period to allow further samples to be taken if required. Sections may be laid out on material suitable for storage in formalin. Annotations can be drawn on the material to indicate the position of the each section and those submitted for processing, then rolled up to retain the pieces in sequential order when returned to the specimen container.

  • Protocols for the communication of special instructions for embedding should be established and well-understood so that microscopic sections demonstrate the tissue in the required plane of dissection.
  • One method commonly used is a dot of ink on the tissue surface to face upward (obverse surface) at embedding.
  • Guidelines on the packaging and transport of specimens should be consulted in establishing protocols on the referral of specimens to external locations. 21
  • Guidelines on the retention of specimens should be consulted in establishing protocols on the release of specimens (e.g. to patients and relatives) and tissue disposal.22
References
  1. National Pathology Accreditation Advisory Council (NPAAC). Requirements for the Performance of Anatomical Pathology Cut-Up. 4th ed. Canberra: Australian Government, Department of Health and Ageing; 2012.
  2. Standards Australia. Medical laboratories -Requirements for quality and competence, AS ISO 15189-2013. SAI Global Limited, Sydney, 2013.
  3. National Pathology Accreditation Advisory Council (NPAAC). Requirements for the Supervsion of Pathology Laboratories. 3rd ed. Canberra: Australian Government, Department of Health and Ageing; 2007.
  4. Royal College of Pathologists of Australasia. Supervision of Diagnostic Laboratories in Australia. Surry Hills, NSW, 2014.
  5. National Pathology Accreditation Advisory Council (NPAAC). Requirements for Medical Pathology Services. Canberra: Australian Government, Department of Health and Ageing; 2013

  6. Makary MA, Epstein J, Pronovost PJ, Millman EA, Hartmann EC and Freischlag JA. Surgical specimen identification errors: a new measure of quality in surgical care. Surgery 2007;141(4):450-455.
  7. Layfield LJ and Anderson GM. Specimen labeling errors in surgical pathology: an 18-month experience. Am J Clin Pathol ;2010;134(3): 466-470.
  8. National Pathology Accreditation Advisory Council (NPAAC). Guidelines for Approved Pathology Collection Centres (Requirements for Medical Pathology Specimen Collection), National Pathology Accreditation Advisory Council, Australian Government, Department of Health and Ageing, Canberra, 2013.
  9. SQHS. Standards fact sheet – Standard 5: Patient identification and procedure matching. Sydney, NSW: Australian Commission on Safety and Quality in Health Care; 2012.

  10. Lester SC. Extraneous Tissue. Manual of Surgical Pathology. 3rd ed. Philadelphia: Saunders Elsevier; 2010. p. 33-4.
  11. Gephardt GN and Zarbo RJ. Extraneous tissue in surgical pathology: a College of American Pathologists Q-Probes study of 275 laboratories. Arch Pathol Lab Med 1996;120(11):1009-1014.
  12. Laslowski A, Savino R. A new approach to minimise the problem of patient to patient contamination in histology. Aust J Med Sci. 2012;33(2):42-7.
  13. Royal College of Pathologists of Australasia (RCPA). The Pathology Request-Test-Report Cycle - Guidelines for Requesters and Pathology Providers. Surry Hills, NSW: RCPA; 2014.
  14. Royal College of Pathologists of Australasia. Biobanking. Surry Hills, NSW, 2014.
  15. Lester SC. Safety Precautions. Manual of Surgical Pathology. 3rd ed. Philadelphia: Saunders Elsevier; 2010. p. 196-203
  16. Suvarna KS, Layton C, Bancroft JD. Bancroft's Theory and Practice of Histological Techniques. 7th ed: Churchill Livingstone; 2012.
  17. Royal College of Pathologists of Australasia. Frozen Section: minimisation of infection hazards associated with cryostats. Surry Hills, NSW, 2014.
  18. Leong AS and Gilham PN. The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology 1989;21(4): 266-268.
  19. Goldstein NS, Ferkowicz M, Odish E, Mani A and Hastah F. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol 2003;120(1):86-92.
  20. Owens SR, Wiehagen L, Simmons C, Sikorova A, Stewart W, Kelly S, Nestler R and Yousem SA. Numerical fidelity of endoscopic biopsy fragments in the processing sequence of a university surgical pathology laboratory. Arch Pathol Lab Med 2011;135(12):1561-1564.
  21. National Pathology Accreditation Advisory Council (NPAAC). Requirements for the Packaging and Transport of Pathology Specimens and Associated Materials, Australian Government, Department of Health and Ageing, Canberra, 2013.
  22. National Pathology Accreditation Advisory Council (NPAAC). Requirements for the Retention of Laboratory Records and Diagnostic Material, Australian Government, Department of Health, Canberra, 2013.