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Flashcards in Monitoring Engraftment Deck (8)
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1
Q

What is HSCT?

A
  • Patients with advanced stage leukaemia or lymphoma may be selected for Haematopoietic Stem Cell Transplantation (HSCT).
  • The criteria that determine whether a patient is eligible for HSCT include general fitness or the presence of other risk factors (e.g. heart disease), severity of the disease, failure of other treatments, the presenco of poor risk prognostic markers.
2
Q

Give some HSCT background.

A
  • Children and younger adults tend to respond well to transplantation with a high rate of recovery following an intense course of chemotherapy plus radiotherapy.
  • Such patients usually receive a myeloablative regimen which is designed to deplete the malignant cells within the bone marrow, peripheral blood, and lymph nodes.
  • Recent advances in transplantation have led to the use of non-myeloablative transplant regimens.
  • This method involves the use of lower doses of chemotherapy and often eliminates the radiotherapy stage all together.
3
Q

Outline Donor lymphocyte infusions.

A
  • To combat the recurrence of the disease, donor T-cells may be administered to the patient post transplant if the disease returns.
  • A finely tuned combination of T-cell donations and immunosuppressant druge allow an effective balance between a potent graft versus leukaemia effect and a manageable graft versus host disease.
4
Q

What is chimerism following HSCT?

A
  • Chimerism is a term used to describe the presence of donor cells within the host.
  • 100% chimerism means that no recipient cells can be detected within the compartment under investigation (e.g. bone marrow).
  • Chimerism assesses engraftment through semi-quantification of DNA extracted from post HSCT patient cells.
  • The chimerism investigation can be carried out on bone marrow, peripheral blood, sub-cellular fractions purified from peripheral blood (e.g. T-cells).
5
Q

Describe the fractionation of peripheral blood for monitoring engraftment.

A
  • Sub-cellular fractionation techniques allow determination of engraftment within various cell types including T-cells, B-cells, NK cells and dendritic cells.
    1) . Antibodies that target specific cell surface antigens are bound to magnetic beads.
    2) . The beads are incubated with the peripheral blood sample and bind to their target cell.
    3) . The sample is decanted and washed in the presence of a magnet which hold the beads and the target cells in the tube.
    4) . Removing the magnet releases the beads and DNA can be extracted from the purified cells.
6
Q

How is chimerism monitored?

A
  • Chimerism is monitored with the use of STR genetic profiling.
  • STR numbers vary between individuals.
  • e.g. Promega PowerPlex 16 kit is an example of a multiplex kit which incorporated primer sets for 16 different loci - this gives a discrimination of around 1 in 10^15 - not as great for chimerism as often related.
7
Q

How is genetic profiling applied in post transplant assessment of chimerism?

A
  • Primary screening involves genetic profiling of the donor and the recipient pre-transplant samples to identify informative alleles.
  • Following analysis, a single marker can be used to measure post transplant engraftment or the multiplex can be run to provide multiple values from which a mean can be derived.
  • Alternative methods include QPCR for single nucleotide or insertion deletion polymorphisms.
  • NGS
  • Calculation - the peak height or area are expressed as arbitrary values and can be used semi-quantitively to calculate the amount of PCR product present.
    European guidelines suggest chimerism be assessed on days 28, 56, 100 post transplant and then every 3 months assuming the patient reaches 100% donor chimerism.
8
Q

Outline different types of HSCT.

A

Sibling allogenic - from sibling

Haplo-allogenic - from a parent or child who shares half of their inherited alleles with the recipient.

Matched unrelated donor (MUD) - Unrelated donor usually from the bone marrow register.

Autologous - patients own cells (harvested when the patient was in remission).

Syngeneic - from an identical twin.

Umbilical cord - stem cells from umbilical cord donations - naive and have very little immuogenicity - this means that they do not have to be as closely genetically matched to the recipient. However, only a small number of progenitors in cord sample. May be overcome by using two cord harvests to increase the number of cells infused into the patient. A second disadvantage is that without a strong immunological presence, the cord donation is less able to fight the leukaemia cells should they return. The presence of 3 DNA profiles following transplantation greatly reduces the potential of identifying multiple informative alleles for chimerism testing. The use of a single allele for post-transplant monitoring is acceptable. The overall cell numbers are low following HSCT.