Chimerism

OVERVIEW

Chimerism analysis is offered for the monitoring of donor marrow engraftment following allogenic bone marrow transplant (BMT).  Prior to transplant, profiles of the donor and the recipient are generated by PCR amplification of fifteen polymorphic short tandem repeat (STR) loci as well as the sex marker Amelogenin (X/Y). Following transplant, these profiles are used to determine the relative proportion of donor and recipient hematopoiesis in the transplant recipient.  In the post-transplant setting, peripheral blood specimens are routinely fractionated prior to analysis, allowing CD3 (lymphoid) and CD33 (myeloid) engraftment to be independently estimated. When clinically indicated, chimerism analysis may also be performed on unfractionated bone marrow aspirates.

INDICATION

  • Pre-transplant assessment: for microsatellite analysis of prospective patient-donor pairs
  • Post-transplant assessment: for monitoring of engraftment in patients following allogenic bone marrow transplant.  Patients are routinely tested at 75 days post-transplant, with follow-up testing in patients with sub-optimal engraftment.

TEST REQUIREMENTS

Pre-transplant

  • Completed CGL Myeloid Testing or CGL Lymphoid Testing requisition form
    • Select “Chimerism: Pre-transplant assessement”
    • Select if the specimen originates from the Donor or the Recipient
    • In the Reason for Testing box, identify the associated recipient or donor
  • Peripheral blood (EDTA) – 1 tube (see Specimen Guidelines)

Post-transplant

  • Completed CGL Myeloid Testing or CGL Lymphoid Testing requisition form
    • Select “Chimerism: Post-transplant assessment”
  • Completed Stem Cell Assay (SCA) requisition form
    • Select “Chimerism Post-transplant assessment”
  • Peripheral blood (Sodium Heparin) – 4 tubes (see Specimen Guidelines)
  • Send specimen and requisition directly to Stem Cell Assay at Terry Fox Laboratory for pre-test fractionation (see Shipping)
    • Do not send post-transplant specimens directly to CGL – fractionation at Stem Cell is required prior to testing
    • Specimens must be received at SCA before 3:30PM Mon-Thurs

For alternate specimen types or other non-routine testing, please contact the laboratory prior to sending a specimen.

TURN-AROUND TIME

  • Pre-transplant: Fourteen calendar days from receipt of specimen and completed, signed requisition form
  • Post-transplant: Seven calendar days form receipt of fractionated specimen and completed, signed requisition form.

RESULTS REPORTING

Pre-transplant: Results are reported as Monitor Possible or Monitor Not Possible along with the number and identity of informative markers between donor and recipient specimens

Post-transplant: Results are reported as the percent engraftment for each of the CD3 lymphoid and CD33 myeloid fractions, calculated as an average of all informative markers within an estimated error range.

METHOD

Analysis is performed on whole peripheral blood (pre-transplant assessment), or CD3 (lymphoid) and CD33 (myeloid) peripheral blood fractions (post-transplant assessment).  A minimum purity of 80% is required for CD3/CD33 blood fractions.  Genomic DNA is extracted from the submitted specimen and kit-based microsatellite analysis of sixteen markers is performed using the PowerPlex 16 HS System according to the manufacturer’s instructions (Promega).

In the pre-transplant setting, the number of informative markers between donor and recipient is determined.  A marker is considered informative if the patient and donor differ in at least one allele. In the post-transplant setting, donor engraftment is determined by comparing the average relative amplification of donor and recipient alleles of all informative microsatellite markers.  Markers included in this kit are D3S1358, THO1, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1P0, PentaD, Amelogenin, VWA, D8S1179, TPOX, and FGA.  Fragment analysis is performed using a 3500 Genetic Analyzer and Genemapper5.0 software.  Quantitative estimates of engraftment are determined using the relative differences in peak area for informative markers and reported as an average of all informative markers.

REFERENCES

Promega Technical Manual PowerPlex 16HS System