MRD testing -
Information for Clinicians

Each sample sent to the MRD Lab should be labelled with a hospital patient sticker (showing full name, DOB, MRN) and accompanied by an Analysis Request Form, indicating type of sample (eg bone marrow, blood, etc) preferably with the same sticker. Either use the specific Study/ALLG form, or a haematology request form, with the MRD, bone marrow sample and specific therapy timepoint clearly written.  

If the diagnosis is not clear at the time of sending the sample, please write “?ALL” and  “?MRD” on the form. Once the diagnosis is known, please email us. This is especially important if MRD is not required so we can avoid significant waste of time and effort and unnecessary charges.

Within a week of diagnosis or relapse, please:

• provide any information that was missing on the request form
• confirm the ALL diagnosis and ALL type (T-ALL or B-ALL) and notify us if a patient has a high-risk translocation involving MLL or KMT2A, ABL1, ABL2, PDGFRB
• provide the names and email addresses of those to receive the MRD report (consultant and CRA)
• email the completed Parent/Patient Consent Form (see links provided below) to mrd@ccia.org.au

Note: To check if a diagnostic or relapse sample has already been sent to the MRD Lab for testing, please check your hospital system or contact us.

Parent/Patient Consent Forms

Currently, there are two different versions of the MRD consent forms – please check which is the approved form at your hospital.

Patient consent form – Response to therapy: MRD testing and research

Parent/Guardian consent form – Response to therapy: MRD testing and research

Patient consent form – MRD testing and banking of specimens for future research

Parent/Guardian consent form – MRD testing and banking of specimens for future research

Reports for MRD results

The results of MRD testing for each patient will be sent to you, the clinician, as a PDF attachment by email to the address(es) provided, within 5 working days of receiving the remission sample. A de-identified copy of the report will also be sent to the Study Centre, for patients enrolled on a clinical trial.

The MRD report will provide the necessary information on sensitivity of the MRD assays and on MRD levels at Timepoint 1 and Timepoint 2 to enable stratification of the patient into risk groups.

Costs

Please contact us at mrd@ccia.org.au for cost enquiries.

Diagnosis samples

The diagnosis sample should be collected in an ACD tube (at least 5 ml) and have a minimum of 1x 10^-7 mononuclear cells after ficoll purification, in order to yield at least 40 ug DNA both for PCR to identify markers and to provide enough DNA for the standard dilution curves used in all subsequent real-time PCR MRD assays.

In a case with a dry tap, a bone marrow trephine sample should be collected and placed in ACD collection tube without formalin. In some patients with high blast cell counts, a peripheral blood sample may be a suitable substitute for bone marrow at diagnosis.

If the disease is present in tumour tissue, a biopsy should be taken and stored in a collection jar or cryovial with a small amount of saline.

Note: Patients with insufficient diagnosis material cannot be followed by PCR MRD.

Remission samples

Marrow aspirates taken in remission should be on the first ‘suck’ aspirate with collection of least 3ml for MRD testing by PCR in ACD tubes, to provide a minimum of 5x10⁶ cells after ficoll purification and yield at least 10 ug DNA to enable molecular MRD assays to be done. 

Peripheral blood cannot be substituted for bone marrow remission samples. If an end-of-induction bone marrow sample is too hypocellular, the aspirate can be repeated 4 days later.

Insufficient samples

If the diagnostic bone marrow or essential trial remission timepoint samples for MRD by PCR have insufficient cells for MRD, the clinician and trial centre will be notified by email.

Relapse samples

Please label request form for any confirmed or suspected relapse samples as “Relapse” or “?Relapse” to ensure proper processing. Some trials require both molecular and flow studies.

Samples for MRD analysis should be sent fresh on the day of collection whenever possible. Please do not delay sending samples because the diagnosis is not clear.

Samples should not be frozen (except in case of delay) and not chilled. Each sample should be sent at ambient temperature on the day of collection, using an overnight courier for centres outside of NSW.

If it is necessary to collect a sample on a Friday, Saturday, Sunday or public holiday, then it should be snap frozen at -80ºC and transported on dry ice the following week. Please ensure sufficient dry ice is included for the duration of shipment.

Please note that deliveries cannot be received after 4pm.

Samples should be sent to:

The MRD Lab
Children’s Cancer Institute
Upper Campus Store, E26 Bioscience South, LG018 Loading Dock
Via Gate 11, Botany St, University of New South Wales
KENSINGTON NSW 2052

An email notification of dispatch of samples for new patients would be appreciated – please email mrd@ccia.org.au

Delivery information – label for sample

The first step in MRD testing is molecular identification of antigen gene rearrangements − these include immunoglobulin and T-cell receptor genes (IG/TCR). PCR and DNA sequencing are used to identify specific antigen receptor gene rearrangements in the leukaemic cells at diagnosis – this usually takes about 3-4 weeks.

Real-time PCR assays with patient-specific primers are then used to detect and quantify these markers of the patient’s leukaemia in remission samples, with sensitivity levels of 10^-4 or greater. The MRD marker in the remission sample is measured relative to a standard curve made from the leukemic reference (e.g. diagnosis).

The steps involved in identification of MRD markers are:

1. cell purification
2. DNA isolation
3. DNA quality control by RQ-PCR on DNA
4. 24 different PCRs to identify several hundred possible rearrangements
5. additional PCRs for T-ALL patients
6. 6 RQ-PCRs to identify generic MRD tests
7. gels and heteroduplex testing
8. DNA sequencing (off site)
9. sequence analysis and primer design
10. primer synthesis (off site)
11. RQ-PCR optimisation.

Assuming the reference sample (diagnosis or relapse) was sent 3-4 weeks earlier, it takes about 3 working days to generate a report. There are several steps from receipt and verification of a sample before the report is sent out.

These steps include:

1. cell purification
2. DNA isolation
3. DNA quantitation
4. DNA quality control by RQ-PCR on DNA
5. setting up patient specific assay RQ-PCR with all the standard dilutions in duplicate, negative controls in 6 and sample in triplicate
6. analysing the data
7. preparing the report.
8. review and sign-off by two authorised staff.

For general enquiries, please contact the MRD Team on 02 7209 6757 or email mrd@ccia.org.au.

Enquiries can also be directed towards our clinical scientist, Dr Michelle Henderson via MHenderson@ccia.org.au and Medical Director of Diagnostics Testing, Dr Toby Trahair via Toby.Trahair@health.nsw.gov.au.

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