1. Minimal Residual Disease (MRD) Detection Technologies
Dr. Nibras Saleam Al-Ammar

2. What is MRD?
Minimal residual disease (MRD) refers to leukemic cells which have survived in the blood of cancer patients during or after treatment.

3. MRD
is a powerful prognostic indicator in newly diagnosed childhood acute lymphoblastic leukemia (ALL).

4. When MRD goes undetected or untreated, it is the primary cause of returning cancer. This has created a need for sensitive MRD tests for cancer diagnosis & research.

5. MRD studies revealed that many patients who achieve morphologic remission harbored residual disease, a finding that is associated with a higher risk of relapse.

6. Investigators of a study group found that patients with MRD levels of 0.1% or higher on both day 33 and day 78 of treatment had a relapse rate of 75%

7. ►Based on the observation that detection of MRD before allogeneic hematopoietic stem cell transplantation (HSCT) is associated with an increase risk of relapse post-HSCT
►MRD assays increasingly are being used to select the timing of transplant.

8. After transplant, detection of MRD can serve as an indicator for
decreasing immunosuppressive therapy
and/or administering donor lymphocyte infusions.

9. Detection of MRD
researchers discovered that the disease was caused by a genetic abnormality
beginning in one cell which then rapidly divides
leaving the harmful genetic sequence in the DNA of all subsequent cells.

10. ■ This led to the development of tests based on DNA ■ These tests were designed to detect the particular DNA sequence linked to the onset of leukemia ■ and use a chain reaction technique to generate cells.

11. FRET
Fluorescence Resonance Energy Transfer (FRET) is a physical phenomenon first described over 50 years ago, that is being used more and more in biomedical research and drug discovery today.

12. ☼FRET relies on the distance-dependent transfer of energy from a donor molecule to an acceptor molecule.
☼ Due to its sensitivity to distance, FRET has been used to investigate molecular interactions.

13. FRET Applications Receptor/ligand interactions
Structure and conformation of proteins
Receptor/ligand interactions
Real-time PCR assays and SNP detection

14. Fluorogenic protease assays
Structure, conformation and hybridization of nucleic acids
Immunoassays
Fluorogenic protease assays

15. Specific Detection using Target Specific Probes
Specific detection of real time PCR is done with some oligonucleotide probes labeled with both a reporter fluorescent dye and a quencher dye.

16. Applications of FRET Probes
Quantitative PCR.
DNA copy number
measurements.
Pathogen detection assays.
SNP genotyping.
Verification of microarray results.

17. TaqMan probes
►TaqMan probes are designed to increase the specificity of Real-time PCR. ►The method was first reported in 1991 by researchers at Cetus Corporation. ► developed by Roche Molecular Diagnostics for diagnostic assays and by Applied Biosystems for research applications.

18. TaqMan probes were named after the videogame PacMan (Taq Polymerase + PacMan = TaqMan) as its mechanism is based on the PacMan principle.

19. Principle
TaqMan probes consist of a fluorophore (FAM) covalently attached to the 5’-end of the oligonucleotide probe and a quencher (TAMRA) at the 3’-end. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via FRET.

29. Thank You