NOTE: Our laboratory offers multiple testing options for APL. Please see our APL overview page.
Droplet digital reverse-transcription polymerase chain reaction (ddRT-PCR) is the method of choice for the measurement of PML-RARA mRNA. A major advantage of RT-ddPCR over QRT-PCR methods of measuring molecular burden is the notion that absolute ratios can be obtained without comparison to a standard curve. Like QRT-PCR, RT-ddPCR allows for monitoring response to therapy over time. RT-ddPCR from peripheral blood (or bone marrow) should be performed before initiation of therapy in order to confirm that the transcript variant present at diagnosis is amenable to monitoring via this method.
This method is only useful for monitoring the P3R3 and P6R3 variants of PML::RARA. All other variants are monitored via standard RT-PCR. Cumulative results over time are reported categorically, qualitatively as positive or negative, as well as a log ratio of [PML::RARA]/[BCR].
1. Reflex test in patients with demonstrated t(15;17) positive disease (diagnosis either by FISH or karyotype) to establish a baseline value
2. Follow-up test for monitoring of minimal residual disease (or measurable residual disease).
The following testing interval applies to follow-up testing:
- Uncomplicated, patients treated with an ATRA/ATO based therapy.
- Every three months. Ending three years post completion of all planned therapy
- At the discretion of the treating physician, testing may be carried out after Induction and each of the consolidation phases.
- Post-transplant patients not being specifically treated for APL.
- Monitoring is at the discretion of the treating physician though it is anticipated that monitoring would not continue indefinitely.
- For any scenario not covered above, please contact the laboratory in order to come to a mutually agreeable testing interval.
Any treating physician
- Completed CGL Myeloid Testing requisition form
- One of the following specimens (please see our Specimen Guidelines, RNA Molecular test type)
- 20mL Peripheral Blood in EDTA tubes (purple top)
- 2.5mL Bone marrow aspirate in EDTA tubes (purple top)
- Peripheral blood derived white blood cells fixed in methanol/acetic acid (residual cytogenetic specimen)
- Bone marrow specimen fixed in methanol/acetic acid (residual cytogenetic specimen)
Note: Results are only quantitatively comparable to previous analyses if the specimen source and type are the same (i.e. fresh peripheral blood to fresh peripheral blood).
Figure 1: Selected requisition entries for RT-ddPCR testing of APL patients.
Total RNA is extracted from the submitted specimen and subjected to RT-ddPCR. The amount of PML-RARA fusion transcript (either P3R3 or P6R3) present in this sample is quantified with respect to BCR transcript (statistical assessment of droplet positivity) and expressed as a log ratio. In order to avoid misinterpretation, the results are also reported qualitatively in the results table as well as categorically (Summary statement).
Figure 2: Detail of dd-RTPCR report
Up to 90% of APL cases will have a translocation involving PML and RARA detectable by this method. At diagnosis, RT-ddPCR positivity is equivalent to the identification of t(15;17) by FISH. Subsequent MRD monitoring will follow the breakpoint identified by this test.
TURN AROUND TIME
Results are reported within ten working days from receipt of specimen and completed requisition form.
Droplet Digital PCR – Applications Guide (Biorad) https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf