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Bone marrow sampling guidance (including MRD)
Guidance on bone marrow aspirate and trephine sampling requirements, including disease-specific pathways and MRD follow-up.
At diagnosis/relapse of acute leukaemia (AML or ALL) or pancytopenia of unknown cause
Taken in order (please note both blue and red tubes in CUH contain EDTA):
Bedside slides for morphology
1–2 mL in EDTA for flow cytometry (1 x red)
2 x 2–3 mL in EDTA tube for molecular testing and DNA/RNA storage (2 x blue tubes)
1–2 mL in EDTA for FISH (suggest blue)
2–3 mL in cytogenetics media for karyotyping
Trephine biopsy is strongly recommended in the following situations:
Aparticulate aspirate
Diagnosis not confirmed from PB flow cytometry, e.g. blasts < 20%
Acute leukaemia of ambiguous lineage, possible acute erythroid leukaemia or megakaryoblastic AML
Possible transformation of previous MPN or CML
A trephine biopsy is extremely unlikely to provide additional relevant information if the diagnosis of AML or ALL is confirmed in the peripheral blood and the conditions above are not met (information on fibrosis or mast cell disorders may be missed without trephine biopsy).
In elderly patients for supportive care with disease in the peripheral blood, a bone marrow aspirate or trephine biopsy may not be required at all.
A trephine biopsy is contraindicated in suspected APML due to the risk of bleeding.
The results of FISH and FLT3/NPM1/IDH1 testing are important when choosing initial treatment. For expediency, please send separate samples for each testing modality to avoid delays from sharing samples between laboratories.
In the event of a “dry tap”, a second trephine biopsy should be collected into a universal container (in 1 mL of sterile saline) for disaggregation in order to perform the above tests. In addition, if circulating blasts, please send 20 mL of PB in EDTA for testing and an additional 10 mL in a cytopot. Discuss with the HODS team.
Managing Challenging Sample Collections
If bone marrow aspiration is unsuccessful (‘dry tap’):
Obtain a second trephine biopsy
Place it in a universal container with 1 mL sterile saline
Label the container 'For cell disaggregation'
Discuss with the HODS team
For patients with circulating blasts, collect:
20 mL peripheral blood in EDTA
An additional 10 mL in a cytopot
Discuss with the HODS team
AML follow-up post-therapy (including MRD)
If no clinical suspicion of relapse (that is, blood counts are stable, patient is asymptomatic, and there is no suspicion of graft failure if post-allograft):
Bedside slides for morphology:
collect < 0.5 mL bone marrow aspirate
2–3 bedside slides are sufficient
put any excess aspirate into the “first pull” EDTA tube
If a molecular marker was detected at diagnosis (this is documented on alert section of EPIC and in the diagnostic sample integrated report):
Collect 2 to 3 mL in EDTA (blue tube) for molecular MRD assessment, label as "first pull"
Collect 2 to 3 mL in EDTA (red tube) for flow cytometry, label as "second pull"
If no molecular marker was detected at diagnosis:
Collect 2 to 3 mL in EDTA (red tube) for flow cytometry, label as "first pull"
FISH can be performed on this sample if relevant
Trephine biopsy:
Perform a trephine biopsy if applicable (see guidelines below)
Minimal Residual Disease (MRD) Testing
Sample Submission Guidelines
Clearly mark all request forms with "FOR MOLECULAR MRD" when submitting samples via the Haematopathology and Oncology Diagnostic Service (HODS).
Specific Testing Instructions
For patients with NPM1-mutated Acute Myeloid Leukaemia (AML) post-cycle 2 of DA-based chemotherapy, send 20ml of peripheral blood for NPM1 quantification. Include this information in the clinical details on the request form.
RNA Extraction Requirements
MRD tests requiring RNA extraction include:
NPM1
BCR::ABL1 (p190)
PML::RARA
CBFB::MYH11
RUNX1::RUNX1T1
Sample Dispatch and Processing
Samples received Monday to Thursday can be dispatched to external laboratories via the Genomic Laboratory Hub (GLH) without processing if received on the same day as collection or by 1.30pm the following day.
Samples received outside these times will need processing at the GLH, leading to delayed turnaround times.
Trephine Biopsy Guidelines
Post-chemotherapy bone marrow assessment
A trephine biopsy is not routinely required if a particulate aspirate is obtained.
If the aspirate is insufficient, review the pre-treatment blast phenotype. CD34-negative blasts are difficult to enumerate on a trephine biopsy, so obtaining a quality aspirate sample is crucial.
Consult with a HODS team member or clinical consultant if you are unsure about sample requirements.
Post-transplant bone marrow assessment
Include a trephine biopsy as part of the "Day +100" post-transplant assessment.
A trephine biopsy is not required with every bone marrow biopsy post-transplant if:
A good or particulate aspirate sample is obtained
Blood counts are stable
There are no clinical concerns of graft failure
ALL follow-up post-therapy (including MRD)
Post phase 1, and subsequent cycles
Post phase 1, and subsequent cycles (note: post phase 1 is a decision point for UKALL2011 protocol, and post course 2 is a decision point for UKALL14 protocol, but sample requirements are the same):
Bedside slides for morphology
2–3 mL in EDTA for IgH /TCR molecular MRD assessment (label first pull).
2–3 mL in EDTA for flow cytometry
Additional 2–3 mL in EDTA for BCR-ABL1 for Ph+ ALL
Trephine biopsy if applicable (see notes below)
Notes
Molecular MRD
All samples for molecular MRD testing that are received via HODS must state “FOR MOLECULAR MRD” on the request form.
MRD tests that require RNA extraction include (but are not limited to) BCR::ABL1 (p190). These can usually be dispatched to external laboratories via the GLH without processing if they are received on Monday-Thursday, either on the same day as collection or if the collection date was the day prior and the sample is received before 1.30pm. Samples not received in this timeframe will require processing in the GLH prior to external dispatch and overall turnaround times will be delayed.
Trephine biopsy
A trephine biopsy is usually not needed for non-transplanted ALL follow up.
Trephine biopsy should be included as part of the “Day +100” post-transplant assessment; however, it is not required with every BM biopsy in the post-transplant setting provided that a good/particulate aspirate sample has been obtained, blood counts are stable, and there are no clinical concerns of graft failure.
Myeloma or Lymphoma
Bedside slides for morphology
2–3 mL red EDTA tube for flow cytometry
2–3 mL red EDTA tube for FISH analysis (Note: Use standard EDTA tube, not Cytopot. Cytopot is only required if unexplained cytopenias are present, e.g., when considering MDS.)
Trephine biopsy (important for assessing disease distribution in myeloma and lymphoma)
Notes
FISH analysis is increasingly valuable at diagnosis. If the initial aspirate sample is aparticulate, consider re-attempting the aspirate to obtain a better quality sample, as this may enhance the likelihood of obtaining a valid FISH result.
MPN (including CML), MDS, or unexplained cytopenia
Bedside slides for morphology
2–3 mL EDTA for flow cytometry
2–3 mL EDTA for FISH if needed
2–3 mL EDTA for molecular testing
5–10 mL in cytogenetics media for karyotyping
Trephine biopsy (often the most diagnostic test so important to obtain an adequate sample) – BUT no trephine biopsy needed in CML if good particulate aspirate obtained
Bone Marrow Biopsy Sample Requirements Summary
| Investigation Type | New/Relapsed Acute Leukaemia or Unexplained Cytopenia | AML follow-up* | ALL follow-up* | Myeloma and Lymphoma | MPN/CML and MDS |
|---|---|---|---|---|---|
| Bedside slides # | 4–5 | 3 | 3 | 3 | 4–5 |
| Flow (EDTA) | Yes (1 tube) | Yes (1 tube) First pull if NO molecular marker | Yes (1 tube) First pull if NO molecular marker | Yes (1 tube) | Yes (1 tube) |
| Molecular testing (EDTA) - including MRD and chimerism | Yes (2 tubes) | Yes First pull if molecular marker | Yes First pull if molecular marker (plus 1 extra tube if Ph+) | Yes (1 tube) | Yes (1 tube) |
| FISH (EDTA) | Yes (1 tube) | Yes (1 tube) | No | Yes (1 tube) | Yes (1 tube) |
| Karyotype (cytopot) | Yes | No | No | No (unless unexplained cytopenia) | Yes (1 tube) |
| Trephine | Yes (unless known/suspected APML) | Not usually needed (see notes) | Not usually needed (see notes) | Yes (important) | Important in MPN, helpful in MDS, not usually needed in CML (see notes) |
| Total EDTAs # | 4 | 2–3 | 2–3 | 3 | 3 |
*if no suspicion of overt disease
Bone marrow aspiration and trephine biopsy: video demonstration
This video demonstrates bone marrow aspiration and trephine biopsy. These procedures are essential for diagnosing and managing many blood disorders.
About this video
Professor George Follows, Consultant Haematologist at Cambridge University Hospitals NHS Foundation Trust, demonstrates these procedures with a patient volunteer. This resource is designed for healthcare professionals to improve their bone marrow sampling techniques and patient care skills.
What this video covers
The demonstration shows how to:
perform bone marrow aspiration and trephine biopsy
inform patients and obtain consent
communicate effectively with patients
collect samples safely and accurately
provide post-procedure care for patients
Useful resources
Standard Operating Procedure (SOP)
For CUH healthcare professionals, an up-to-date SOP for bone marrow aspiration and trephine biopsy is available on Oncolnet:
Document: "Bone marrow aspiration and/or trephine biopsy" (OH/B005)
Location: Cancer Directorate Document Library
Patient information leaflet
A printable leaflet is available about bone marrow aspiration and trephine biopsy procedures. You can share this with patients to support your discussions.
The leaflet includes:
reasons for the procedure
what happens during the test
possible risks
aftercare advice
View the CUH bone marrow aspiration and trephine biopsy patient information leaflet (↗)