Monitoring of Hematopoietic Chimerism by Short Tandem Repeats, and the Effect of CD Selection on Its Sensitivity

Abstract

The assessment of chimerism, the ratio of donor to recipient cells, is critical for monitoring the engraftment of donor cells and determining the recurrence of the original disease after allogeneic bone marrow transplantation (BMT). Chimerism in patients after BMT has been assessed by several methods (1). Fluorescence in situ hybridization (FISH) analysis is particularly well established (1). Although FISH analysis is a highly sensitive and quantitative method, its application is restricted to cases with sex-mismatched BMT because the sex chromosome is the common chimerism marker (1). Many minisatellites, such as short tandem repeats (STRs), are highly polymorphic because of allelic variation in repeat copy numbers (2)(3). A method that makes use of this polymorphism, PCR-based analysis of STRs (PCR-STR), which is a useful tool for human identification in forensic testing (4), has been used to monitor hematopoietic chimerism after BMT (5)(6)(7)(8)(9)(10)(11)(12)(13). In recent years, a method using real-time quantitative PCR has been developed (14). This method is more sensitive than PCR-STR, but it requires special instrumentation and expensive reagents and thus is unsuitable for routine assay in the clinical laboratory. We investigated the assay characteristics of PCR-STR by comparing it with FISH analysis and examining the effect of cell selection (according to the immunophenotype of the original leukemic clone) on the ability of PCR-STR to detect chimerism. Our institutional ethics committee approved this study, and participants gave informed consent before participation. DNA was extracted from whole peripheral blood taken from two unrelated healthy volunteers (male and female), and from the fraction containing mononuclear cells and granulocytes, with use of DNA extractor WB (Wako Pure Chemicals). The repeat regions of six STR loci were identically amplified by PCR (Table 1⇓ ). …