Chimersim

CHIMERISM TESTING FOR ENGRAFTMENT ANALYSIS

Chimerism testing (engraftment analysis) is performed for patients who have received a hematopoietic stem cell transplant. The test involves identifying the genetic profiles of the recipient and the donor and then evaluating the extent of mixture in the recipient’s blood or bone marrow. First, DNA is isolated from the recipient and potential donor before the transplant and analysis is performed to determine whether the genetic markers unique to the donor and the recipient have sufficient power to distinguish the donor from the recipient. Next, after the transplant takes place, the performance of the transplant engraftment is assessed by evaluating the donor versus recipient contribution of white blood cells in post-transplant blood or bone marrow specimens obtained from the recipient.

• The DNA is isolated from the recipient, donor, and post-transplant samples.
• DNA is amplified via the Promega GenePrint24 System which employs methodology commonly used in human identity testing and is accomplished by the analysis of genomic polymorphisms called short tandem repeat (STR) loci.
• The percent donor Chimerism is calculated and reported, and a longitudinal plot of historic data is generated for successive post-transplant specimens.

CLINICAL UTILITY 

Important clinical events in allogeneic bone marrow transplantation such as engraftment, relapse, and the effects of post-transplant therapies can be monitored on a molecular level by detecting genetic differences between recipient and donor, to help guide clinical decision making (Bader et al, 2005; Lion et al,  2012; Clark et al, 2014).

Criteria for Patient Testing 

The intended use of this assay is for routine post-transplant documentation of the donor/recipient origin of white blood cells in peripheral blood and/or bone marrow. Additional clinical indications include:

• Test for risks of graft rejection and recurrence of disease.
• Document presence of donor cells in post-transplant patient with residual disease or prior to donor lymphocyte infusion (DLI).
• Evaluate donor/recipient cells in patients with inadequate marrow function.
• Determine if malignancy is a recurrence or new occurrence from donor cells.
• Differentiate donor cell populations in recipients who have received multiple transplants.

Specimen Requirements 

Test: Pre-transplant analysis, donor

Specimen type: Whole blood, minimum 2 mL in lavender (EDTA) or yellow (ACD) collection tube. Package and ship specimen cold, but not frozen.

Test: Pre-transplant analysis, recipient

Specimen type: Whole blood, minimum 2 mL in lavender (EDTA) or yellow (ACD) collection tube OR DNA*. Package and ship specimen cold, but not frozen.
Alternatively: 2 buccal brushes** each in a 2 mL tube with 650 uL of Lysis Buffer (only if engraftment has already occurred).

Test: Post-transplant Chimerism testing

Specimen type: Whole blood, minimum 2 mL OR bone marrow, minimum 1 mL in lavender (EDTA) or yellow (ACD) collection tube OR DNA*. Package and ship specimen cold, but not frozen.

Remarks: Post-transplantation results will be compared to pre-transplant recipient and donor genotypes, therefore, donor and recipient specimens should be obtained and genotyped before the transplant event occurs.

*DNA samples should be received only if they are the only samples available. DNA specimens are only accepted if the nucleic acid isolation occurred in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS. Such samples will be processed however, a successful outcome may not be guaranteed.

**A buccal swab alternative source of DNA may only be used if there is an acceptable confidence level to determine the recipient’s genotype.

Requisition

All orders need to be accompanied with a requisition (via FAX or Secure email). Contact AZClinCore for additional information.

Turnaround Time

Report delivered within 5 working days from receipt of the tissue sample(s) by AZClinCore, based on business hours of the laboratory: Monday through Friday, 9am-5pm Arizona Time. STAT services are available; contact the laboratory for more information.

Methodology and Additional Details

Chimerism testing (engraftment analysis) by DNA employs methodology commonly used in human identity testing and is accomplished by the analysis of genomic polymorphisms called short tandem repeat (STR) loci. These loci consist of a core DNA sequence that is repeated a variable number of times within a discrete genetic locus. The term STR, also referred to as a microsatellite, relates to the number of base pairs of a tandemly repeated core DNA sequence which ranges from 2-8 base pairs in length. These loci exhibit alleles that may differ in length between individuals and are inherited as codominant Mendelian traits.

The GenePrint 24 System is a 24-locus multiplex system designed to generate a multi-locus human DNA profile from a variety of human-derived biological sources. This five-color system allows co-amplification and fluorescent detection of the following autosomal STR loci: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D10S1248, D22S1045, D2S441, D1S1656, D12S391, D2S1338, D19S433, Penta D and Penta E plus Amelogenin for gender determination. In addition, the male-specific DYS391 locus is included to identify null Y allele results for Amelogenin.

Data analysis is facilitated by fragment analysis software (ChimerMarker) which sizes the DNA fragments using an internal lane standard run with the sample and assigns genotypes by comparison to an STR allelic ladder. This provides distinct STR genotypic profiles for the donor and for the transplant recipient. STR loci that are polymorphic (i.e. informative) between these individuals are used to assess relative amounts of recipient and donor DNA in the post-transplant sample.

References:

1. Bader P1, Niethammer D, Willasch A, Kreyenberg H, Klingebiel T. How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplant. 2005 Jan;35(2):107-19.
2. Lion T1, Watzinger F, Preuner S, Kreyenberg H, Tilanus M, de Weger R, van Loon J, de Vries L, Cavé H, Acquaviva C, Lawler M, Crampe M, Serra A, Saglio B, Colnaghi F, Biondi A, van Dongen JJ, van der Burg M, Gonzalez M, Alcoceba M, Barbany G, Hermanson M, Roosnek E, Steward C, Harvey J, Frommlet F, Bader P. The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation. Leukemia. 2012 Aug;26(8):1821-8. doi: 10.1038/leu.2012.66. Epub 2012 Mar 7.
3. Clark JR1, Scott SD, Jack AL, Lee H, Mason J, Carter GI, Pearce L, Jackson T, Clouston H, Sproul A, Keen L, Molloy K, Folarin N, Whitby L, Snowden JA, Reilly JT, Barnett D; United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group. Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group. Br J Haematol. 2015 Jan;168(1):26-37. doi: 10.1111/bjh.13073. Epub 2014 Aug 22.