35 Bone Marrow Transplant Engraftment

Question 1

What is HSCT?

Answer 1

Hematopoietic stem cell transplantation

Question 2

What inherited disorders can be treated with HSCT?

Answer 2

hemoglobinopathies;

Bone marrow failure syndromes;

immunodeficiencies;

inborn metabolic diseases

Question 3

What acquired disorders can be treated with HSCT?

Answer 3

acquired hematological malignancies;

solid tumors

Question 4

What are the sources of hematopoietic stem cells?

Answer 4

peripheral blood (obtained by apheresis) - can be advantageous;

bone marrow - traditional source;

cord blood

Question 5

What is the advantage of T-cell depletion prior to HSCT?

Answer 5

minimize the risk of graft-versus-host disease (GVHD)

Question 6

What are the disadvantages of T-cell depletion prior to HSCT?

Answer 6

increases the risk of graft rejection;

diminishes graft-versus-leukemia (GVL) activity

Question 7

What test used to monitor engraftment of HSCT?

Answer 7

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

Question 8

What are short tandem repeats (STRs)?

Answer 8

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

Question 9

Why STRs are used for identity testing

Answer 9

The number of times any given STR is repeated is highly polymorphic between individuals

Question 10

Specimen used for monitoring HSCT engraftment

Answer 10

DNA extracted from leukocytes present in peripheral blood and bone marrow

Question 11

How does the multiplex PCR kit determine the relative contribution of cells from donor and recipient?

Answer 11

1. Primer target STRs;
2. PCR amplification –> resultant fluorescently-labeled PCR products;
3. Capillary gel electrophoresis;
4. Calculations of the peak aera or peak height to determine the relative contribution of cells from donor and recipient

Question 12

What is the stutter during STRs PCR amplification?

Answer 12

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

Question 13

What is the common stutter?

Answer 13

one repeat smaller

Question 14

If the donor and recipient share an allele that differs by only a single repeat, can we use this donor?

Answer 14

No;
The common stutter is one repeat smaller, this will make it hard to differentiate the donor from recipient cell.

Question 15

What is chimerism testing (engraftment analysis) for HSCT?

Answer 15

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

Question 16

What is the sensitivity of chimerism testing (engraftment analysis) of STRs for HSCT?

Answer 16

1-5%

Question 17

When using HSCT as the treatment of malignant disease, persistent or increasing recipient cells means what?

Answer 17

relapse or survival of host hematopoietic cells

Question 18

What cells are most frequently performed for chimerism studies in HSCT?

Answer 18

CD3+ cells;

increase or high levels of recipient CD3+ T cells
and CD56+ NK cells are associated with graft rejection

Question 19

Increase or high levels of what cells are associated with graft rejection in HSCT?

Answer 19

recipient CD3+ T cells and CD56+ NK cells

Question 20

What is the greatest limitation of chimerism testing?

Answer 20

sensitivity