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Chapter 141 : Detecting Chimerism after Blood and Marrow Transplantation
This chapter discusses fundamental aspects of laboratory assessment of hematopoietic chimerism after blood and marrow transplantation. Clinical indications for chimerism testing in blood and marrow transplantation include posttransplant monitoring of engraftment kinetics and stable mixed chimerism as well as detecting relapse and graft loss. Additional applications include detecting cells engrafted from a third party and distinguishing monozygotic and dizygotic twins. Chimerism testing is routinely used after allogeneic blood and marrow transplantation to monitor the status of the allograft and to detect relapse. In the early years of bone marrow transplantation, chimerism testing was primarily limited to use for monitoring donor engraftment. In 2005, the predominant methods for chimerism testing involve amplification of short tandem repeats (STR) loci. There are many situations in which there is coexistence of donor and host hematopoietic cells, and this phenomenon is referred to as mixed chimerism. In patients with mixed chimerism, the proportion (percentage) of donor cells can change, and this condition is described as increasing mixed chimerism if the percentage of donor cells is increasing or decreasing mixed chimerism if the percentage of donor cells is decreasing. For blood and marrow transplantation, chimerism analysis has become an important component of posttransplant monitoring. In addition to the clinical indications, chimerism testing can be used to test for the presence of third-party cells derived from blood transfusions, maternal cell engraftment in immunocompromised children, and maternal cell contamination of umbilical cord blood units.
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STR alleles. Two STR alleles that are distinguished by the numbers of tandem repeats of the sequence motif AAAG are depicted at the top of the figure. The solid black sections represent conserved sequences which are located in the flanking regions of the STR. Oligonucleotides labeled 5’ P and 3’ P anneal to these flanking regions to selectively prime DNA synthesis in the PCR. In this example, the 5’ P primer is labeled with a fluorescent dye molecule. The products of the PCR can be separated using slab gel electrophoresis (bottom left), and the fluorescence is often displayed as an electropherogram (bottom right).
Electropherograms for a donor-recipient pair. Electropherograms for multiplex reactions for four loci are shown. For D21S11, the donor and recipient are homozygous for the same allele (223 bp). For D18S51, the recipient is homozygous for an allele with 285 bp. The donor shares this allele and has an additional informative allele (302 bp). For D16S539, the donor and recipient share the 364-bp allele, and there are informative alleles for the donor (360 bp) and recipient (352 bp). For Penta D, there are two informative recipient alleles (422 and 441 bp) and two informative donor alleles (397 and 417 bp).
Admixtures of control DNA for a donor-recipient pair. The electropherogram for the homozygous recipient is shown in the top panel. The small peak to the immediate left of the main peak is a stutter peak. The panel labeled “100% Donor” shows the alleles from the control DNA from the donor. The small peak to the immediate left of the two large peaks is a stutter peak. Presumably, the stutter peak from the larger of the donor alleles comigrates with the smaller allele. The next panels show the amplification products of admixtures of donor and recipient DNA. Arrows show the host peak in the admixtures.
Testing of cell subsets to increase sensitivity. The two upper left panels show the electropherograms of the host alleles and donor alleles. The host allele is undetectable in electrophero-grams for peripheral blood, CD14/15, and CD19 subsets using the standard scale and an expanded scale (right). For the CD3 subset, the host allele is barely detectable in the standard scale but is clearly evident in an expanded scale. Subset analysis not only provides increased sensitivity but also shows the lineage of the autologous cells.