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At diagnosis/relapse of acute leukaemia (AML or ALL) or pancytopenia of unknown cause
AML follow-up post-therapy (including MRD)
ALL follow-up post-therapy (including MRD)
MPN (including CML) and MDS or unexplained cytopenia
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
Flow cytometry:
2–3 mL in EDTA tube (red tube)
Molecular testing:
2 x 3–5 mL in EDTA tubes (2 blue tubes); one for in-house testing and one for MRD marker detection (if ALL)
FISH analysis:
2–3 mL in EDTA (blue tube)
Karyotyping:
2–5 mL in cytogenetics media (cytopot)
Trephine biopsy:
Recommended in all cases except if known or suspected acute promyelocytic leukaemia (APML)
Expediting Test Results
FISH and FLT3/NPM1 testing are essential for treatment decisions in acute leukaemia. To ensure timely results, submit separate samples for FISH and FLT3/NPM1 tests. This prevents delays from sample sharing between laboratories.
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
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)
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.
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
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.
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.
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
| 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
This video demonstrates bone marrow aspiration and trephine biopsy. These procedures are essential for diagnosing and managing many blood disorders.
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.
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
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
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 (↗)