Bone biopsies, non-tumour bone and synovium

Background

A range of specimens containing bone may be received in the laboratory.

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Bone marrow trephines are frequently undertaken to investigate haematological malignancies such as lymphoma, leukaemia and multiple myeloma. 1,2 Core biopsies, open surgical bone biopsies and closed percutaneous bone biopsies are undertaken to investigate a range of bone diseases. 3,4 Other small bone specimens include bone curettings and intervertebral disc material from surgery to repair herniated discs containing some bone. 3 Larger specimens of bone include femoral head removed during hip replacement surgery and occasionally incidental rib specimens removed during procedures such as thoracotomy. 5

This protocol is applicable to bone biopsies (including bone marrow trephines), non-tumour bone specimens, intervertebral disc, synovium and other miscellaneous soft tissue specimens. See separate protocols for bone tumour, soft tissue tumour and femoral head specimens.


Record the patient identifying information and any clinical information supplied together with the specimen description as designated on the container. See overview page for more detail on identification principles.

Fresh tissue received
  • No
    • Non-routine fixation (not formalin), describe.
  • Yes
    • Special studies required, describe.
    • Ensure samples are taken prior to fixation.
Intraoperative consultation
  • Not performed
  • Performed, describe type and result
    • Frozen section
    • Imprints
    • Other, describe

See general information for more detail on specimen handling procedures.

Inspect the specimen and dictate a macroscopic description.

External InspectionBack to top


Orientate and identify the anatomical features of the specimen.

Orientation markers

Record additional orientation or designation provided by operating clinician:

  • Absent
  • Present
    • Method of designation (e.g. suture, incision)
    • Featured denoted

Photograph the intact specimen if required.

Describe the following features of the specimen:

Procedure

Record as stated by the clinician.

Options
  • Synovial biopsy, describe
  • Bone marrow trephine
  • Core biopsy of bone
  • Bone curettings
  • Intervertebral disc
  • Rib from thoracotomy
  • Meniscectomy
  • Other, describe

Anatomical components included (more than one may apply) and specimen dimensions (mm)

Describe and measure the anatomical components present.
  • Number of cores/pieces
  • Total specimen size
    • Bone marrow trephine, aggregate length of cores and specify if adequate/inadequate for examination1
    • Bone core biopsy in two dimensions
    • Other specimens in three dimensions
  • Bone, specify (i.e. which bone submitted)
    • Without articular cartilage
    • With articular cartilage
  • Synovium
  • Meniscus
  • Other, describe

Specimen integrity

  • Intact
  • Disrupted/fragmented, describe

Specimen appearance

  • Colour
  • Crystalline/chalky deposits
    • Absent
    • Present –if fresh, sample with needle, smear onto slide for microscopic examination under polarised light 4

DissectionBack to top


Synovial biopsies

Before sectioning or submitting the specimen it is important to try to orientate the specimen so that sections can be taken perpendicular to the synovial surface which has a roughened or frond-like appearance.4 The aim is to include the full thickness of the synovium and underlying connective tissue.

Synovial specimens taken from joint replacement operations may contain large pieces of metallic debris which should be avoided when sampling as the debris may interfere with sectioning.

In any setting where crystal deposition disease is suspected, formalin fixation, subsequent decalcification and haematoxylin staining are likely to dissolve crystalline structures. In such cases a scrape smear (prior to processing) of any chalky substance examined under polarised light is optimal.4,5

  • Specimens <5mm do not require any dissection and can be submitted in their entirety. Painting the deep aspect with ink may facilitate embedding perpendicular to the synovial surface.
  • Serially section larger specimens transversely at 3-4mm intervals perpendicular to the synovial surface.
Bone marrow trephines (BMT)

BMT do not require dissection. Allow to fix for a minimum of three hours, wash thoroughly before transferring for decalcification in appropriate solution. Overnight treatment in an acid or chelating decalcification solution is generally sufficient. See decalcification for more information.

Bone core biopsies

Core biopsies generally do not require dissection (unless >5mm in thickness when they may be divided). 4 Allow to fix for a minimum of three hours 4, wash thoroughly before transferring for decalcification in appropriate solution.6-8

Overnight treatment in an acid or chelating decalcification solution is generally sufficient. Consideration of the possibility of future FISH studies should be undertaken depending on the clinical setting. If deemed likely, decalcification in EDTA may be prudent.5

Bone curettings and other small bony specimens

Intervertebral disc and curetted specimens generally do not require dissection.

Allow to fix overnight 4, wash thoroughly before transferring for decalcification in appropriate solution.6-8 Any chalky material (possible crystalline material) should be smeared  on to a slide and examined with polarisation microscopy prior to processing. 4,5 Hard parts of the specimen may be separated from soft areas of tissue to allow for longer decalcification if required. 4

Bone resection non-tumour specimens

Large specimens containing bone are slow to fix and require initial dissection prior to fixation and decalcification.6 See separate decalcification protocol for more information.

Various saws are available for the dissection of bone; hand saws, band saws and/or diamond-coated saws. Selection of appropriate equipment will depend on the resources in a particular laboratory.6

Dissect any soft tissue tumour and submit for processing.

Section specimen with an appropriate bone saw according to diagrams provided.

In small non-tumour sections that include articular bone, sections should ensure that the relationship of the cartilage to subchondral bone is clearly demonstrable.

In fragmented specimens, representative fragments of bone and soft tissue plus anything chalky or necrotic (dense or white) should be submitted.

Loose bodies should include the central area as this usually incorporates the precipitating event or nidus.

Rib from thoracotomy

Dissect with an appropriate bone saw, cutting 20mm transverse sections. Allow to fix sufficiently, gently brush the surface to remove bone dust and wash thoroughly before transferring for decalcification in appropriate solution.

After sectioning the specimen may require longer fixation in larger quantity of formalin.

Photograph the dissected specimen, if required.

Note photographs taken, diagrams recorded and markings used for identification.

Internal InspectionBack to top


Synovial small biopsies, BMT, bone cores, curettings and other small specimens do not require further description.

For synovial excision specimens, the presence of any tears should be commented on in the macroscopic description.

ProcessingBack to top


See general information for more detail on decalcification principles.

Synovial small biopsies

Submit all tissue transferred directly into cassettes for processing. Lens paper, biopsy pads or similar are required to prevent loss of tissue during processing.

Sections may be processed through alcohol to optimise crystal identification. 

If the tissue is received in formalin, it is best processed within 12 hours.

After sectioning, an eosin-only or unstained sections may prove useful as haematoxylin dissolves crystals. 5

Synovial excision and resection specimens

Synovial excision specimens including intra- and para-articular supporting tissues

  • Submit a representative section of each heterogeneous area of the specimen.
  • Menisci and labrum of hip, submit radial sections including both the peripheral capsular rim and inner aspect.
  • Tendons and ligaments, submit longitudinal sections to best demonstrate abnormalities.
Bone core biopsies

After decalcification, wash thoroughly and transfer entire specimen directly to cassettes for processing. Lens paper, biopsy pads or similar lining in cassettes are required to prevent loss of tissue during processing.

Bone curettings and other small bony specimens

After decalcification, wash thoroughly and transfer entire specimen directly to cassettes for processing.

Where large quantities of curetted material is present, submit at least one block per 10mm. Biopsy pads or similar lining in cassettes are required to prevent loss of tissue during processing.

Rib from thoracotomy

Submit a representative section.

Record details of each cassette.

An illustrated block key similar to those provided below may be useful.

Block allocation key

Synovial biopsies, BMT, bone cores and curettings
Cassette id
Site
No. of pieces
A
All tissue submitted
 
Synovial excision and resection specimens
Cassette id
 
No. of pieces
A-B
Heterogeneous areas, representative section of each
 
C
Tendon and ligament, if present, abnormalities, longitudinal sections
 
D
Articular bone, if present, sections demonstrating relationship of cartilage to bone
 
E-F
As applicable: fragmented specimens, representative sections of bone, soft tissue, chalky or necrotic areas as applicable. Loose bodies, sections from central area.
 
Rib from thoracotomy
Cassette id
Site
No. of pieces
A
One representative section
 

Acknowledgements

Associate Professor Fiona Bonar for her contribution in reviewing and editing this protocol.

ReferencesBack to top


  1. Lindeman R, Juneja S, Maxwell DE, Erber W, Lee S-H, Knottenbelt E, et al. Bone Marrow Specimen (Aspirate and Trephine Biopsy) Structured Reporting Protocol. Royal College of Pathologists Australasia, Surry Hills, NSW, 2014.
  2. Ramsay A, Attygale A, Menon G, Naresh K and Wilkins B. Tissue pathways for lymph node, spleen and bone marrow trephine biopsy specimens, The Royal College of Pathologists, London, 2008.
  3. Athanasou NA and Mangham DC. Dataset for histopathology reports on primary bone tumours, The Royal College of Pathologists, London, 2010.
  4. Freemont AJ, Denton J and Mangham DC. Tissue pathways for bone and soft tissue pathology, The Royal College of Pathologists, London, 2011.
  5. Lester SC. Manual of Surgical Pathology, Saunders Elsevier, Philadelphia, 2010.
  6. Dimenstein IB. Bone grossing techniques: helpful hints and procedures. Ann Diagn Pathol 2008;12(3):191-198.
  7. Bancroft JD and Gamble M. Theory and practice of histological techniques. Churchill Livingstone Elsevier, Philadelphia, PA 2008.
  8. Suvarna KS, Layton C and Bancroft JD. Bancroft's Theory and Practice of Histological Techniques. Churchill Livingstone, 2012.