A Chimera was a creature in Greek mythology usually represented as a composite of a lion, goat, and serpent. Contemporary use of the term “chimerism” in hematopoietic cell transplant derives from this idea of a “mixed” entity, referring to someone who has received a transplant of genetically different tissue. A test for chimerism after a hematopoietic stem cell transplant involves identifying the genetic profiles of the recipient and of the donor and then evaluating the extent of mixture in the recipient’s blood, bone marrow, or other tissue.
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 microsatellites, 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. STR loci have been identified throughout the human genome and some loci have more than 25 alleles.
DNA sequence information within the conserved flanking regions of the loci is used to create oligonucleotide primer pairs for the STRs. These primers are used in PCR (polymerase chain reaction) amplification of test samples. This technique can amplify the STR sequence as many as a billion times, providing material that can be separated with an electrophoretic gel or by capillary electrophoresis (CE). Genotyping is done by evaluation of the DNA fragment sizes. Reference to an allelic ladder may be used for exact identification of STR alleles.
The PCR-based STR/CE system has several advantages over other methods of analysis. The amplification of multiple STR loci can be combined (multiplexed) in a single tube, permitting analysis of up to sixteen loci in one reaction. Since minute amounts of DNA are required, samples with low cell numbers can be used, and the small size of the STR alleles even makes it possible to use degraded DNA samples. The digital data facilitates analysis and archiving, and the CE process is both fast and cost effective. PCR amplification and analysis of STR loci provides a rapid and reliable method for the evaluation of engraftment status in the stem cell transplantation setting.
Currently used technology allows the co-amplification and three-color detection of sixteen loci which are subdivided into 3 sets of 5 or 6 loci that exhibit amplified fragments with non-overlapping size ranges.
During the PCR amplification step, the amplified fragments are labeled with fluorescent dyes. After PCR amplification, the samples are processed on a capillary electrophoresis (CE) system.
Data analysis is facilitated by a fragment analysis software which sizes the DNA fragments using an internal lane standard run with the sample and assigns genotypes by comparison to an STR allele ladder included in the CE run. 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.
Samples tested may be from any material containing DNA, including bone marrow, peripheral blood, solid tumors, epidermal tissue, hair follicles, buccal swabs, and fractionated cell subsets. Since PCR amplification of a sample is routinely performed with less than 2 ng of genomic DNA (equivalent to approximately 300 cells), chimerism testing by this method can be successfully performed even for patients with graft failure, severe leukopenia, or from hematopoietic cell subset fractions. STR analysis has been used to evaluate the engraftment status of patients who have received a hematopoietic cell transplant including patients receiving double cord blood donor units or a second transplant from a different donor; as well as to confirm the genetic identity of putative identical twins, and to detect in-utero derived maternal cell engraftment among patients with a diagnosis of Severe Combined Immunodeficiency (SCID). STR/CE analysis is a rapid, reliable, accurate and reproducible procedure.
Limitations of the Assay
- Sufficient DNA must be isolated from the test samples to allow robust PCR amplification. DNA isolation methods must be available to handle low cell count samples, such as might be encountered in recipients with graft failure and from Flow Cytometry lineage specific sorted white blood cell subsets. Samples may have concentrations too low to quantify by UV Spectrophotometry OD260. DNA samples from 5,000 to 30,000 isolated cells reliably provide acceptable amplification. The laboratory has guidelines that define when sample analysis should be repeated.
- With the commercial kits, many STR loci have informative alleles for both donor and recipient in the unrelated donor setting. However, the number of informative STR loci may be limited to as few as 3 among related donor transplant pairs. Quantitative values representing the mean of as few as 3 STR loci have been found to be reproducible.
- Patients with malignant diseases may have clonal mutations that impact certain STR loci. A patient allele may be absent at one or more STR loci when comparing the alleles identified in the patient pre-transplant sample versus the post-transplant sample. In rare circumstances, an extra patient STR allele may be detected at a specific locus that was not present in the pre-transplant sample. In most cases these anomalies are likely due to chromosome translocations. Missing alleles could also be the result of a mutation within the STR conserved flanking sequences where the PCR primers are located. The data from STR loci with missing or extra alleles is not used for quantification.
- If patient pre-transplant cells are not available, samples such as buccal swabs, skin biopsy or hair roots may be collected post-transplant. However, it should be noted that buccal samples may contain significant amounts of donor cells.
- The assay is not intended for the detection of minimal residual disease.
Clinical Indications for Chimerism Testing in Hematopoietic Cell Transplant
Routine post-transplant documentation of the donor/recipient origin of white blood cells in peripheral blood and/or marrow. Documentation of engraftment may include testing lineage-specific cell subsets, such as CD3 positive T-cells and CD33 positive myeloid cells.
Evaluate donor/recipient cells in patients with inadequate marrow function.
Define whether recurrent or new malignancy has originated from recipient or donor cells.
Assess prognostic risks of rejection and recurrent malignancy.
Document the persistence of donor cells post-transplant in patients with recurrent disease or prior to donor lymphocyte infusion (DLI).
Evaluate whether graft rejection has occurred in recipients that are candidates for a second transplant.
Differentiate the origin of donor cells in recipients who have received a second transplant with a different donor or a transplant with double cord blood units.
Detect the presence of maternal derived cells in patients diagnosed with Severe Combined Immuno-Deficiency (SCID).
Verify genetic identity of putative identical twins.
Frequently Asked Questions
Question: How are multiple donors analyzed?
Answer: It is essential to identify STR loci that show at least one unique STR allele for the patient and each donor.
Question: Why is maternal engraftment tested for?
Answer: Patients diagnosed with a Severe Combined ImmunoDeficiency (SCID) may have become engrafted with hematopoietic cells of maternal origin in-utero. Certain lineage specific cell subsets (especially CD3-positive cells) may be predominantly or entirely of maternal origin.
Question: What is the difference between “full” and “mixed” chimerism?
Answer: “Full” chimerism is used to refer to a patient which exhibits a post-transplant phenotype in hematopoietic cells that is all of donor origin.
“Mixed” chimerism refers to a patient which exhibits a mixture of patient and donor phenotype in hematopoietic cells after transplant.
Question: What if a recipient baseline sample is not available?
Answer: Several options are available for obtaining a recipient sample post-transplant: buccal wipe, hair root, or skin biopsy samples can be used for a recipient baseline sample. Buccal wipe samples should be used with caution since donor cells may be present in significant quantities in buccal samples.
Question: What if a donor baseline sample is not available?
Answer: If the donor is living, obtain a new blood sample. If the donor is not living, post-transplant samples should be compared with the patient pre-transplant baseline to identify STR alleles that are detected but are not of patient origin.
Question: Why is HLA not a feasible means to monitor engraftment?
Answer: Transplant donors are chosen to be as closely matched to the recipient as possible. There are no HLA markers differentiating the recipient and the donor when they are matched, and only one or two if they are mismatched. Additionally, other technologies are often more sensitive for this purpose than monitoring HLA mismatched alleles; consequently, other loci are used to provide unique profiles.
Question: What is an informative STR locus?
Answer: This is a locus with at least one allele unique to the recipient or the donor. To be useful for chimerism calculations, a locus must have alleles unique to both. There may be a large number of informative loci when an unrelated donor is used for the transplant, but very few if a matched sibling is used. Because of differences in amplification efficiencies of the alleles at each locus, it is preferable to get mean or median values from several loci to enhance accuracy. Results from individual loci are reliably reproducible, so even one locus can be used for trending sequential samples.
Question: What is the “amelogenin” locus that is included in some amplification panels?
Answer: The amelogenin gene is located on the X and Y chromosomes. It is not an STR, but displays different sizes of products on the respective chromosomes. This makes it useful for determining gender. It is included in the primer panel by commercial kit vendors targeting the forensics community where X/Y differentiation is used extensively. It is generally not used for chimerism analysis since it can’t provide a unique female marker (a male sample always includes an X allele); nor is it helpful when evaluating samples after a sex-matched stem cell transplant.
Question: How is STR chimerism analysis different from genotyping?
Answer: “Genotype” refers to the particular alleles present in a specified locus. Genotyping is done to establish recipient and donor profiles in chimerism testing. When chimerism analysis is done after a stem cell transplant, the genotypic profiles of the recipient and of the donor are compared to the post transplant sample profile to evaluate how much of each component is present.
Genotyping is also used in other scenarios such as forensics and parentage testing. Forensic analysts use genotypes to identify the source of evidence in criminal cases and to exclude people from consideration as suspects. They also can identify human remains in “John Doe” cases and in mass disasters. Parentage determinations are done in a similar fashion to exclude someone as a possible parent by finding alleles present in the child that don’t match either the mother or the putative father.
Literature Cited/Recommended Reading
Bryant E, Martin PJ. Documentation of engraftment and characterization of chimerism following hematopoietic cell transplantation. In: Forman S, Blune K, Thomas ED, eds. Hematopoietic cell transplantation. 2nd ed. Malden, MA: Blackwell Science, 1998.
Bultler J. Forensic DNA typing. Elsevier Academic Press.