35 Bone Marrow Transplant Engraftment Flashcards
What is HSCT?
Hematopoietic stem cell transplantation
What inherited disorders can be treated with HSCT?
hemoglobinopathies;
Bone marrow failure syndromes;
immunodeficiencies;
inborn metabolic diseases
What acquired disorders can be treated with HSCT?
acquired hematological malignancies;
solid tumors
What are the sources of hematopoietic stem cells?
peripheral blood (obtained by apheresis) - can be advantageous;
bone marrow - traditional source;
cord blood
What is the advantage of T-cell depletion prior to HSCT?
minimize the risk of graft-versus-host disease (GVHD)
What are the disadvantages of T-cell depletion prior to HSCT?
increases the risk of graft rejection;
diminishes graft-versus-leukemia (GVL) activity
What test used to monitor engraftment of HSCT?
identity testing, aka. DNA fingerprinting:
1) short tandem repeats (STRs), aka microsatellites;
2) pyrosequencing SNPs;
3) allele-specific qPCR;
4) TaqMan qPCR;
5) allele-specific PCR of insertion/deletion markers
What are short tandem repeats (STRs)?
1) aka microsatellites;
2) STRs are repetitive DNA sequences of 1-6 bp in length that are repeated in tandem;
3) STRs are about 3% of the human genome;
4) STRs are scattered throughout the genome;
5) Majority STRs are located in noncoding regions;
6) 8% of STRs are in coding regions
Why STRs are used for identity testing
The number of times any given STR is repeated is highly polymorphic between individuals
Specimen used for monitoring HSCT engraftment
DNA extracted from leukocytes present in peripheral blood and bone marrow
How does the multiplex PCR kit determine the relative contribution of cells from donor and recipient?
- Primer target STRs;
- PCR amplification –> resultant fluorescently-labeled PCR products;
- Capillary gel electrophoresis;
- Calculations of the peak aera or peak height to determine the relative contribution of cells from donor and recipient
What is the stutter during STRs PCR amplification?
1) The repetitive nature of STRs causes some slippage of polymerase, resulting smaller or larger product than the true allele.
2) stutters could be <10% of the PCR products
3) common stutter is one repeat smaller
What is the common stutter?
one repeat smaller
If the donor and recipient share an allele that differs by only a single repeat, can we use this donor?
No;
The common stutter is one repeat smaller, this will make it hard to differentiate the donor from recipient cell.
What is chimerism testing (engraftment analysis) for HSCT?
1) Multiple informative markers are analyzed for each posttransplant sample and averaged;
2) Results are reported as % cells of donor origin and % cells of recipient origin
What is the sensitivity of chimerism testing (engraftment analysis) of STRs for HSCT?
1-5%
When using HSCT as the treatment of malignant disease, persistent or increasing recipient cells means what?
relapse or survival of host hematopoietic cells
What cells are most frequently performed for chimerism studies in HSCT?
CD3+ cells;
increase or high levels of recipient CD3+ T cells
and CD56+ NK cells are associated with graft rejection
Increase or high levels of what cells are associated with graft rejection in HSCT?
recipient CD3+ T cells and CD56+ NK cells
What is the greatest limitation of chimerism testing?
sensitivity
